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Checkpoint Controls and Cancer

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M E T H O D S I N M O L E C U L A R B I O L O G Y™
Checkpoint Controls
and Cancer
Volume 1
Reviews and Model Systems
Edited by
Axel H. Schönthal,PhD
Department of Molecular Microbiology and Immunology
and K. Norris Jr. Comprehensive Cancer Center,
University of Southern California Keck School of Medicine,
Los Angeles, CA

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v
Preface
Intracellular checkpoint controls constitute a network of signal transduc-
tion pathways that protect cells from external stresses and internal errors. Exter-
nal stresses can be generated by the continuous assault of DNA-damaging agents,
such as environmental mutagens, ultraviolet (UV) light, ionizing radiation, or
the reactive oxygen species that can arise during normal cellular metabolism. In
response to any of these assaults on the integrity of the genome, the activation of
the network of checkpoint control pathways can lead to diverse cellular responses,
such as cell cycle arrest, DNA repair, or elimination of the cell by cell death
(apoptosis) if the damage cannot be repaired. Moreover, internal errors can
occur during the highly orchestrated replication of the cellular genome and its
distribution into daughter cells. Here, the temporal order of these cell cycle events
must be strictly enforced—for example, to ensure that DNA replication is com-
plete and occurs only once before cell division, or to monitor mitotic spindle
assembly, and to prevent exit from mitosis until chromosome segregation has
been completed. Thus, well functioning checkpoint mechanisms are central to
the maintenance of genomic integrity and the basic viability of cells and, there-
fore, are essential for proper development and survival.
The importance of proper functioning of checkpoints becomes plainly
obvious under conditions in which this control network malfunctions and fails.
Depending on the severity and timing, failure of this machinery can lead to
embryonic lethality, genetic diseases, and cancer. Cancer in particular has been
recognized as a disease in which acquired mutations and loss of genomic integ-
rity decidedly contribute to its origination and progression. Most, if not all,
cancer cells exhibit incomplete or malfunctioning checkpoint control pathways,
which constitutes a situation that is further aggravated because this absence of
efficient controls allows for even more deleterious mutations to accumulate.
The enhanced potential of cancer cells to survive under suboptimal conditions
and their increasing ability to withstand chemotherapeutic intervention are but
two of the consequences. Thus, identifying the molecular components of check-
point controls and understanding the complexity of their spaciotemporal inter-
actions is a major goal of current cancer research. Besides satisfying our academic
curiosity, novel insights and advances in this complex area are essential for the
development of new and more effective therapies.
Experts from 10 different countries have contributed their detailed knowl-
edge to the present two-volume work, Checkpoint Controls and Cancer, which

vi Preface
presents a collection of indispensable tools and their applications that will
further advance our understanding of the intricacies of checkpoint controls.
Each volume is divided into two parts. Part I of Volume 1: Reviews and Model
Systemscontains comprehensive review articles that introduce all of the
important components of checkpoint controls, describe their intricate interac-
tions, and highlight the relevance of these processes to the cancer problem.
Here, the amazing complexities of checkpoint controls—and the gaps that exist
in our knowledge thereof—become distinctly apparent. Part II illustrates the
advantages of utilizing diverse model systems, such as intact human skin or
knockout mice, as well as other most useful organisms, such as Xenopus,Droso-
phila, Caenorhabditis, and yeast. As has been shown time and again, the con-
vergence of information from various model systems is able to crossfertilize
and accelerate research both across disciplines and beyond the boundaries of a
particular species. This is especially true for the study of yeast, which has
already provided major insights into the function of cell cycle and checkpoint
controls.Volume 2: Activation and Regulation Protocolsis a collection of
“how-to” chapters with stepwise instructions that focuses on the individual
components of checkpoint controls and describes the detailed analysis of their
activity. Part II completes this collection by providing various experimental
approaches for the manipulation of checkpoint pathways and the analysis of
the resulting consequences for the cellular phenotype. Altogether, this collec-
tion of protocols and proven techniques will be useful for all researchers,
whether they be novices who need step-by-step instructions, or experienced
scientists who want to explore new approaches or model systems for the study
of checkpoint controls and cancer.
I would like to thank the authors for the clear and detailed descriptions
of the procedures they provided, and for the many useful hints they included
in the notes section to each chapter. Those authors who provided general
review chapters are thanked for their thoughtful and nicely structured presen-
tation of their topics and some marvelous illustrations. I wish to acknowledge
John Walker, the series editor, for his support, and the staff at Humana Press
who helped produce this volume.
Axel H. Schönthal

vii
Contents of Volume 1
Reviews and Model Systems
Preface..............................................................................................................v
Contents of the Companion Volume................................................................ix
Contributors.....................................................................................................xi
P
ARTI. REVIEWS OF CHECKPOINT CONTROLS, THEIR INVOLVEMENT IN THE DEVELOPMENT
OF
CANCER,AND APPROACHES TO THEIR INVESTIGATION
1 G1 and S-Phase Checkpoints, Chromosome Instability,
and Cancer
Hiroshi Nojima..................................................................................... 3
2 Analyzing the G2/M Checkpoint
George R. Stark and William R. Taylor.............................................. 51
3 Analyzing the Spindle Checkpoint in Yeast and Frogs
P. Todd Stukenberg and Daniel J. Burke............................................ 83
4 Cell Cycle Checkpoint Control Mechanisms
That Can Be Disrupted in Cancer
Bipin C. Dash and Wafik El-Deiry...................................................... 99
PART II. ANALYZING CHECKPOINT CONTROLS IN DIVERSE MODEL SYSTEMS
5 Establishment of a Cell-Free System to Study the Activation
of Chk2
Xingzhi Xu and David F. Stern.......................................................... 165
6 Analyzing Checkpoint Controls in Human Skin
Sandra Pavey and Brian G. Gabrielli................................................ 175
7 Generation and Analysis of Brca1 Conditional Knockout Mice
Chu-Xia Deng and Xiaoling Xu......................................................... 185
8 Analysis of Cell Cycle Progression and Genomic Integrity
in Early Lethal Knockouts
Eric J. Brown..................................................................................... 201
9Xenopus Cell-Free Extracts to Study the DNA Damage Response
Vincenzo Costanzo, Kirsten Robertson, and Jean Gautier............... 213

viii Contents
10 A Xenopus Cell-Free System for Functional Analysis
of the Chfr Ubiquitin Ligase Involved in Control
of Mitotic Entry
Dongmin Kang, Jim Wong, and Guowei Fang.................................. 229
11 Control of Mitotic Entry After DNA Damage in Drosophila
Burnley Jaklevic, Amanda Purdy, and Tin Tin Su............................. 245
12 Methods for Analyzing Checkpoint Responses
in
Caenorhabditis elegans
Anton Gartner, Amy J. MacQueen, and Anne M. Villeneuve.......... 257
13 Assaying the Spindle Checkpoint in the Budding Yeast
Saccharomyces cerevisiae
Christopher M. Yellman and Daniel J. Burke................................... 275
14 Purification and Analysis of Checkpoint Protein Complexes
From
Saccharomyces cerevisiae
Catherine M. Green and Noel F. Lowndes....................................... 291
Index............................................................................................................ 307

ix
CONTENTS OF THE COMPANION VOLUME
Volume 2: Activation and Regulation Protocols
PART I. PROTOCOLS FOR THE STUDY OF CHECKPOINT-REGULATORY COMPONENTS
1 Analysis of RB Action in DNA Damage Checkpoint Response
Christopher N. Mayhew, Emily E. Bosco, David A. Solomon,
Erik S. Knudsen, and Steven P. Angus
2 Interaction Between the Retinoblastoma Protein and Protein
Phosphatase 1 During the Cell Cycle
Norbert Berndt and John W. Ludlow
3 Generation of p53 Target Database Via Integration of Microarray
and Global p53 DNA-Binding Site Analysis
Suxing Liu, Asra Mirza, and Luquan Wang
4 Functional Analysis of CDK Inhibitor p21
WAF1
Rati Fotedar, Mourad Bendjennat, and Arun Fotedar
5 Analysis of p21
CDKN1A
Recruitment to DNA Excision Repair Foci
in the UV-Induced DNA Damage Response
Lucia A. Stivala and Ennio Prosperi
6 Quantitative Determination of p16 Gene Expression by RT-PCR
Sylke Schneider, Kazumi Uchida, Dennis Salonga, Ji Min Yochim,
Kathleen D. Danenberg, and Peter V. Danenberg
7 Measuring Cyclin-Dependent Kinase Activity
Axel H. Schönthal
8 Determination of the Catalytic Activities of mTOR and Other Members
of the Phosphoinositide-3-Kinase-Related Kinase Family
Gary G. Chiang and Robert T. Abraham
9 CHK1 Kinase Activity Assay
Ya Wang and Hongyan Wang
10 Assaying Cdc25 Phosphatase Activity
Ingo Hassepass and Ingrid Hoffmann
11 Analyzing the Regulation and Function of ATM
Martin F. Lavin, Shaun P. Scott, Sergei Kozlov, and Nuri Gueven
12 Use of siRNA to Study the Function of MDC1 in DNA Damage
Responses
Zhenkun Lou and Junjie Chen

x Contents of the Companion Volume
13 Functional Analysis of APC-Cdh1
Tamotsu Sudo, Naoto T. Ueno, and Hideyuki Saya
14 Purification of Mitotic Checkpoint Complex, an Inhibitor
of the APC/C From HeLa Cells
Valery Sudakin and Tim J. Yen
15 Analysis of the Spindle-Assembly Checkpoint in HeLa Cells
Paul R. Andreassen, Dimitrios A. Skoufias, and Robert L. Margolis
16 Functional Analysis of the Spindle-Checkpoint Proteins
Using an In Vitro Ubiquitination Assay
Zhanyun Tang and Hongtao Yu
PART II. STUDYING CONSEQUENCES OF CHECKPOINT PATHWAY ACTIVATION
17 Analysis of Checkpoint Responses to Histone Deacetylase Inhibitors
Heather Beamish, Robyn Warrener, and Brian G. Gabrielli
18 Biochemical Analysis of the Cell Cycle and Cell Cycle Checkpoints
in Transiently Transfected Cells After Collection With Magnetic
Beads
Xiaofen Ye, Maxim Poustovoitov, Hidelita Santos, David M. Nelson,
and Peter D. Adams
19 Analysis of DNA Repair and Chromatin Assembly In Vitro
Using Immobilized Damaged DNA Substrates
Jill A. Mello, Jonathan G. Moggs, and Geneviève Almouzni
20 Analyzing Cell Cycle Checkpoints After Ionizing Radiation
Bo Xu and Michael B. Kastan
21 FACS-Based Detection of Phosphorylated Histone H3 for the
Quantitation of Mitotic Cells
William R. Taylor
22 Analysis of Cell Cycle by Flow Cytometry
Piotr Pozarowski and Zbigniew Darzynkiewicz
23 Analyzing Markers of Apoptosis In Vitro
Stéphanie Plenchette, Rodolphe Filomenko, Emmanuelle Logette,
Stéphanie Solier, Nelly Buron, Séverine Cathelin, and Eric Solary
24 Analysis of Telomerase Activity and Telomere Function in Cancer
Katrina E. Gordon and E. Kenneth Parkinson

Contributors
ROBERT T. ABRAHAM • Program in Signal Transduction Research,
The Burnham Institute, La Jolla, CA
P
ETER D. ADAMS • Department of Basic Science, Fox Chase Cancer Center,
Philadelphia, PA
G
ENEVIÈVE ALMOUZNI • Research Section, Institut Curie, Paris, France
P
AUL R. ANDREASSEN • Institut de Biologie Structurale J.-P. Ebel (CEA-
CNRS-UJF), Grenoble, France
S
TEVEN P. ANGUS • Department of Molecular Genetics and Microbiology,
Duke University Medical Center, Durham, NC
H
EATHER BEAMISH • Centre for Immunology and Cancer Research, University
of Queensland, Princess Alexandra Hospital, Brisbane, Queensland,
Australia
M
OURAD BENDJENNAT • Institut de Biologie Structurale J.-P. Ebel (CEA-
CNRS-UJF), Grenoble, France
N
ORBERT BERNDT • Division of Hematology/Oncology, Children’s Hospital
Los Angeles, Los Angeles, CA
E
MILY E. BOSCO • Department of Cell Biology, Vontz Center for Molecular
Studies, University of Cincinnati College of Medicine, Cincinnati, OH
E
RIC J. BROWN • Department of Cancer Biology, Abramson Family Cancer
Research Institute, University of Pennsylvania School of Medicine,
Philadelphia, PA
D
ANIEL J. BURKE • Department of Biochemistry and Molecular Genetics,
University of Virginia Medical Center, Charlottesville, VA
N
ELLY BURON • INSERM U517, IFR100, Faculty of Medicine, Dijon, France
S
ÉVERINE CATHELIN • INSERM U517, IFR100, Faculty of Medicine, Dijon,
France
J
UNJIE CHEN • Department of Oncology, Mayo Clinic, Rochester, MN
G
ARY G. CHIANG • Program in Signal Transduction Research, The Burnham
Institute, La Jolla, CA
V
INCENZO COSTANZO • Department of Genetics and Development, Columbia
University, New York, NY
K
ATHLEEN D. DANENBERG • Response Genetics Inc., Los Angeles, CA
P
ETER V. DANENBERG • Department of Molecular Biology and Biochemistry
and K. Norris Jr. Comprehensive Cancer Center, University of Southern
California, Keck School of Medicine, Los Angeles, CA
xi

ZBIGNIEW DARZYNKIEWICZ • Brander Cancer Research Institute at New York
Medical Center, Hawthorne, NY
B
IPIN C. DASH • Laboratory of Molecular Oncology and Cell Cycle
Regulation, Howard Hughes Medical Institute, and Departments
of Medicine, Genetics, and Pharmacology and Abramson Cancer Center,
University of Pennsylvania School of Medicine, Philadelphia, PA
C
HU-XIA DENG • Genetics of Development and Disease Branch, Digestive
and Kidney Diseases, National Cancer Institute, National Institutes
of Health, Bethesda, MD
WAFIK EL-DEIRY • Laboratory of Molecular Oncology and Cell Cycle
Regulation, Howard Hughes Medical Institute, and Departments
of Medicine, Genetics, and Pharmacology and Abramson
Cancer Center, University of Pennsylvania School of Medicine,
Philadelphia, PA
GUOWEI FANG • Department of Biological Sciences, Stanford University,
Stanford, CA
R
ODOLPHE FILOMENKO • INSERM U517, IFR100, Faculty of Medicine, Dijon,
France
A
RUN FOTEDAR • Sidney Kimmel Cancer Center, San Diego, CA
R
ATI FOTEDAR • Institut de Biologie Structurale J.-P. Ebel (CEA-CNRS-UJF),
Grenoble, France
B
RIAN G. GABRIELLI • Centre for Immunology and Cancer Research,
University of Queensland, Princess Alexandra Hospital, Brisbane,
Queensland, Australia
A
NTON GARTNER • Department of Cell Biology, Max Planck Institute
for Biochemistry, Martinsried, Germany
J
EAN GAUTIER • Department of Genetics and Development, Columbia
University, New York, NY
K
ATRINA E. GORDON • Beatson Institute for Cancer Research, Glasgow,
Scotland
C
ATHERINE M. GREEN • Genome Damage and Stability Centre, University
of Sussex, Brighton, UK
N
URI GUEVEN • The Queensland Institute of Medical Research, PO Royal
Brisbane Hospital, Herston, Queensland, Australia
I
NGO HASSEPASS • Cell Cycle Control and Carcinogenesis, German Cancer
Research Center (DKFZ), Heidelberg, Germany
I
NGRID HOFFMAN • Department of Applied Tumorvirology, Cell Cycle Control
and Carcinogenesis (F045), German Cancer Research Center (DKFZ),
Heidelberg, Germany
xii Contributors

Contributors xiii
BURNLEY JAKLEVIC • Molecular, Cellular, and Developmental Biology,
University of Colorado, Boulder, CO
D
ONGMIN KANG • Department of Biological Sciences, Stanford University,
Stanford, CA
M
ICHAEL B. KASTAN • Department of Hematology-Oncology, St. Jude
Children’s Research Hospital, Memphis, TN
E
RIK S. KNUDSEN • Department of Cell Biology, Vontz Center for Molecular
Studies, University of Cincinnati College of Medicine, Cincinnati, OH
S
ERGEI KOZLOV • The Queensland Institute of Medical Research, Royal
Brisbane Hospital, Herston, Queensland, Australia
M
ARTIN F. LAVIN • The Queensland Cancer Fund Research Unit,
The Queensland Institute of Medical Research, Royal Brisbane Hospital,
Herston, Queensland, Australia
S
UXING LIU • Tumor Biology Department, Schering-Plough Research
Institute, Kenilworth, NJ
E
MMANUELLE LOGETTE • INSERM U517, IFR100, Faculty of Medicine, Dijon,
France
Z
HENKUN LOU • Department of Oncology, Mayo Clinic, Rochester, MN
N
OEL F. LOWNDES • Department of Biochemistry and National Centre
for Biomedical Engineering Science, National University of Ireland
Galway, Galway, Ireland
J
OHN W. LUDLOW • Vesta Therapeutics, Research Triangle Park, NC
A
MY J. MACQUEEN • Department of Molecular Biology, UT Southwestern
Medical Center, Dallas, TX
R
OBERT L. MARGOLIS • Institut de Biologie Structurale J.-P. Ebel (CEA-
CNRS-UJF), Grenoble, France
C
HRISTOPHER N. MAYHEW • Department of Cell Biology, Vontz Center
for Molecular Studies, University of Cincinnati College of Medicine,
Cincinnati, OH
J
ILL A. MELLO • Research Section, Institut Curie, UMR218 du Centre
National de la Recherche Scientifique (CNRS), Paris, France
A
SRA MIRZA • Tumor Biology Department, Schering-Plough Research
Institute, Kenilworth, NJ
J
ONATHAN G. MOGGS • Research Section, Institut Curie, UMR218 du Centre
National de la Recherche Scientifique (CNRS), Paris, France
D
AVID M. NELSON • Department of Basic Science, Fox Chase Cancer Center,
Philadelphia, PA
H
IROSHI NOJIMA • Department of Molecular Genetics, Research Institute
for Microbial Diseases, Osaka University, Suita, Osaka, Japan

xiv Contributors
xiv Contributors
E. KENNETH PARKINSON • Beatson Institute for Cancer Research, Garscube
Estate, Bearsden, Glasgow, Scotland
S
ANDRA PAVEY • Queensland Institute of Medical Research, PO Royal
Brisbane Hospital, Brisbane, Queensland, Australia
S
TÉPHANIE PLENCHETTE • INSERM U517, IFR100, Faculty of Medicine, Dijon,
France
M
AXIM POUSTOVOITOV • Department of Basic Science, Fox Chase Cancer
Center, Philadelphia, PA
P
IOTR POZAROWSKI • Department of Clinical Immunology, School
of Medicine, Lublin, Poland
E
NNIO PROSPERI • Istituto di Genetica Molecolare del CNR, sez. Istochimica e
Citometria, Pavia, Italy
A
MANDA PURDY • Molecular, Cellular, and Developmental Biology,
University of Colorado, Boulder, CO
K
IRSTEN ROBERTSON • Department of Genetics and Development, Columbia
University, New York, NY
D
ENNIS SALONGA • Response Genetics Inc., Los Angeles, CA
H
IDELITA SANTOS • Department of Basic Science, Fox Chase Cancer Center,
Philadelphia, PA
H
IDEYUKI SAYA • Department of Tumor Genetics and Biology, Graduate
School of Medical Sciences, Kumamoto University, Honjo, Kumamoto,
Japan
S
YLKE SCHNEIDER • Department of Molecular Biology and Biochemistry and
K. Norris Jr. Comprehensive Cancer Center, University of Southern
California Keck School of Medicine, Los Angeles, CA
A
XEL H. SCHÖNTHAL • Department of Molecular Microbiology and
Immunology and K. Norris Jr. Comprehensive Cancer Center, University
of Southern California Keck School of Medicine, Los Angeles, CA
S
HAUN P. SCOTT • The Queensland Institute of Medical Research, Royal
Brisbane Hospital, Herston, Queensland, Australia
D
IMITRIOS A. SKOUFIAS • Institut de Biologie Structurale J.-P. Ebel (CEA-
CNRS-UJF), Grenoble, France
E
RIC SOLARY • INSERM U517, IFR100, Faculty of Medicine, Dijon, France
S
TÉPHANIE SOLIER • INSERM U517, IFR100, Faculty of Medicine, Dijon,
France
D
AVID A. SOLOMON • Department of Cell Biology, Vontz Center for Molecular
Studies, University of Cincinnati College of Medicine, Cincinnati, OH
G
EORGE R. STARK • Department of Molecular Biology, Lerner Research
Institute, The Cleveland Clinic Foundation, Cleveland, OH

Contributors xv
DAVID F. STERN • Department of Pathology, School of Medicine, Yale
University, New Haven, CT
L
UCIA A. STIVALA • Dipartimento di Medicina Sperimentale, sez. Patologia
Generale “C. Golgi,” Università di Pavia, Pavia, Italy
P. T
ODD STUKENBERG • Department of Biochemistry and Molecular Genetics,
University of Virginia Medical Center, Charlottesville, VA
T
IN TIN SU • Molecular, Cellular, and Developmental Biology, University
of Colorado, Boulder, CO
V
ALERY SUDAKIN • Institute for Cancer Research, The Fox Chase Cancer
Center, Philadelphia, PA
TAMOTSU SUDO • Department of Tumor Genetics and Biology, Graduate School
of Medical Sciences, Kumamoto University, Honjo, Kumamoto, Japan
ZHANYUN TANG • Department of Pharmacology, UT Southwestern Medical
Center, Dallas, TX
W
ILLIAM R. TAYLOR • Department of Biological Sciences, University
of Toledo, Toledo, OH
K
AZUMI UCHIDA • Department of Molecular Biology and Biochemistry and K.
Norris Jr. Comprehensive Cancer Center, University of Southern
California Keck School of Medicine, Los Angeles, CA
N
AOTO T. UENO • Department of Blood and Marrow Transplantation,
The University of Texas M. D. Anderson Cancer Center, Houston, TX
A
NNE M. VILLENEUVE • Departments of Developmental Biology and Genetics,
Stanford University School of Medicine, Stanford, CA
H
ONGYAN WANG • Department of Radiation Oncology, Kimmel Cancer Center
of Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA
L
UQUAN WANG • Discovery Technology Department, Schering-Plough
Research Institute, Kenilworth, NJ
YA WANG • Department of Radiation Oncology, Kimmel Cancer Center of
Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA
ROBYN WARRENER • Centre for Immunology and Cancer Research, University
of Queensland, Princess Alexandra Hospital, Brisbane, Queensland,
Australia
J
IM WONG • Department of Biological Sciences, Stanford University,
Stanford, CA
B
O XU • Department of Genetics and Stanley S. Scott Cancer Center,
Louisiana State University Health Sciences Center, New Orleans, LA
X
IAOLING XU • Genetics of Development and Disease Branch, National
Institute of Diabetes, Digestive and Kidney Diseases, National Cancer
Institute, National Institutes of Health, Bethesda, MD

XINGZHI XU • Department of Pathology, Yale University School of Medicine,
New Haven, CT
X
IAFEN YE • Department of Basic Science, Fox Chase Cancer Center,
Philadelphia, PA
C
HRISTOPHER M. YELLMAN • Department of Biochemistry and Molecular
Genetics, University of Virginia Medical Center, Charlottesville, VA
T
IM J. YEN • Institute for Cancer Research, The Fox Chase Cancer Center,
Philadelphia, PA
J
I MIN YOCHIM • Department of Molecular Biology and Biochemistry and K.
Norris Jr. Comprehensive Cancer Center, University of Southern
California, Keck School of Medicine, Los Angeles, CA
H
ONGTAO YU • Department of Pharmacology, UT Southwestern Medical
Center, Dallas, TX
xvi Contributors

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 1
I
REVIEWS OF CHECKPOINT CONTROLS,
T
HEIR INVOLVEMENT IN THE DEVELOPMENT
OF
CANCER,AND APPROACHES TO THEIR
INVESTIGATION

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 3
3
From:Methods in Molecular Biology, vol. 280:Checkpoint Controls and Cancer, Volume 1:
Reviews and Model Systems
Edited by: Axel H. Schönthal © Humana Press Inc., Totowa, NJ
1
G1 and S-Phase Checkpoints, Chromosome Instability,
and Cancer
Hiroshi Nojima
Summary
Mitogen-dependent progression through the first gap phase (G1) of the mammalian cell-
division cycle is precisely regulated so that normal cell division is coordinated with cell
growth, while the initiation of DNA synthesis (S phase) is precisely ordered to prevent
inappropriate amplification of the DNA that may cause genome instability. To ensure that
these fundamental requirements of cell division are met, cells have developed a surveillance
mechanism based on an intricate network of protein kinase signaling pathways that lead to
several different types of checkpoints. Since these checkpoints are central to the mainte-
nance of the genomic integrity and basic viability of the cells, defects in these pathways
may result in either tumorigenesis or apoptosis, depending on the severity and nature of the
defects. This review summarizes the genetic and molecular mechanisms of checkpoint acti-
vation in the G1/S and S phases of the mammalian cell cycle that monitor DNA damage and
replication. The relevance of these mechanisms to the origin of cancer is also discussed.
Key Words: Cell cycle; checkpoints; G1/S; DNA damage; chromosome; p53; MYC; CHK1;
CHK2; ATM; ATR; pRB; E2F; p21; INK4a; CDK2; Cyclin E; Cyclin G; NBS1; MRE11;
BRCA1; MDM2; ARF; Cdc25A.
1. Introduction
Eukaryotic cells have developed a complex network of cell cycle check-
point pathways. These act as surveillance mechanisms that ensure the proper
progression of the cell cycle after exposure to various environmental stresses
or after the occurrence of spontaneous perturbations such as DNA damage and
improper progression of DNA replication (1–4). These evolutionally conserved
surveillance mechanisms ensure that DNA replication remains faithful, thus
guaranteeing the transmission of an unaltered genome and promoting the sur-
vival of the cells. The checkpoint regulatory machineries that serve as the
guardians of proper cell cycle progression fulfill four fundamental consecutive

4 Nojima
tasks. First, upon stress, they rapidly induce cell cycle arrest or delay. They
then help activate the mechanisms that repair damaged DNA or stalled replica-
tion. They also maintain the cell cycle arrest until repair is complete. At this
point, they then actively re-initiate cell cycle progression. The cell cycle arrest
that is induced and maintained by these checkpoints gives the cell time not
only to repair the cellular damage but also to wait for the dissipation of an
exogenous cellular stress signal or to probe the availability of essential growth
factors, hormones, or nutrients.
The cell cycle checkpoints were initially defined as constituting a regulatory
mechanism that acts to arrest the cell cycle in response to DNA damage, so that
cell cycle progression and repair could be temporally coordinated (5,6). How-
ever, more recent work suggests that the checkpoints may play many more
regulatory roles in various cellular events (6). Indeed, they are now believed to
regulate the transcription of DNA damage response genes (7), the telomere
length and chromatin structure (8), the recruitment of proteins to damage sites
(2,3), the kinetochore attachment to spindle microtubules (9), the arrangement
of the cytoskeleton (10,11), meiotic recombination (12,13), meiotic chromo-
some pairing and segregation (14), and the cell cycle timing in the first cell
divisions of the embryo (15). Notably, the checkpoint signaling pathways can
also result in the activation of programmed cell death if cellular damage cannot
be properly repaired (16–19) .
The stability of the genome is under constant threat from chemicals, radia-
tion, and normal DNA metabolism. Therefore, if the checkpoints are not prop-
erly controlled, the cells may suffer potentially catastrophic DNA damage that
can lead to elevated mutation rates, chromosome instability, and aneuploidy,
all of which can contribute to tumorigenesis (20). Failure of the G1/S phase
and S-phase checkpoints to act properly is particularly deleterious because it
may directly elicit chromosomal aberrations and the accumulation of deleteri-
ous mutations, which increase the likelihood of the occurrence of genetic syn-
dromes and diseases such as cancer (21,4). In this review, I will discuss the
recent progress in the study of the mammalian checkpoints at the G1 and S
phases that guard the entry into and the progression through the S phase and
thereby ensure proper DNA replication. Occasionally studies on budding yeast,
fission yeast, and other eukaryotes will be mentioned if their observations aid
our understanding of mammalian checkpoint mechanisms. I will also focus on
the evidence that supports the notion that aberrant checkpoint regulatory mecha-
nisms may promote the incidence of DNA alterations that may lead to cancer.

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 5
2. Molecular Mechanism Controlling the G1/S Transition
of Mammalian Cells
2.1. Factors Regulating G1 Phase of the Cell Cycle
2.1.1. G1 Phase CDK/Cyclin Complexes
The progression through the cell cycle is governed by the periodic activa-
tion and inactivation of cyclin-dependent kinase (CDK) complexes. The CDK
proteins are Ser/Thr protein kinases, and their kinase activities are controlled
by their association partners, which are called cyclins (22). The protein levels
of the CDKs remain constant through the cell cycle, whereas the levels of the
cyclins vary during the cell cycle, owing to periodic expression and degrada-
tion. The timely regulation of different CDK/cyclin complexes is responsible
for well-organized cell cycle progression, as these complexes act in G1 to ini-
tiate S phase and in G2 to initiate mitosis. These mechanisms are conserved
from yeast to mammals (23). The kinase activity of the CDKs is also tightly
controlled by the binding of inhibitors and phosphorylation events.
In the middle of the cyclin proteins is a domain of well-conserved amino
acid sequences called the cyclin box. While cyclins were originally character-
ized as being the regulatory subunit of CDK that is periodically expressed and
degraded during the cell cycle (24), it was later found that many cyclins do not
cycle and that they can regulate cellular functions other than the cell cycle
(25). These include: cyclin G (26), which is a regulatory subunit of protein
phosphatase 1A (27); cyclin H, which forms a complex with CDK7 that regu-
lates not only other CDKs as a CDK-activating kinase (CAK) (28)but also
transcription and DNA repair (29); cyclin L, which is a regulatory subunit of
CDK11 and promotes pre-mRNA splicing (30); and cyclin T, which forms a
complex with CDK9 and activates transcription by hyperphosphorylation of
the carboxyl-terminal domain of the large subunit of RNA polymerase II (31).
Eleven CDK proteins (Cdc2 = CDK1, CDK2, . . . CDK11) have been dis-
covered and examined in mammalian cells to date. Of these, CDK2, CDK3,
CDK4, and CDK6 are principally responsible for G1 progression and entry
into S phase. CDK4 and CDK6 are activated in mid G1, whereas CDK2 is
activated in late G1. While CDK4 and CDK6 are co-expressed in many cell
types, CDK6 does not fully compensate for the function of CDK4 in most cells
(32,33). The CDK4/cyclin D and CDK 6/cyclin D complexes play pivotal
roles in early to mid G1, whereas CDK2/cyclin E and possibly CDK2/cyclin A
function at the late stage of G1 (22). These cyclins are comprehensively termed

6 Nojima
the G1 cyclins (34). Three types of cyclin Ds (D1, D2, and D3) have been
identified, each of which functions as a regulatory subunit of either CDK4 or
CDK6(35). In mid G1 phase, the CDK4/cyclin D complexes phosphorylate
the pRB (retinoblastoma) family of nuclear phosphoproteins (seeSubheading
2.1.4.), which are the key regulators of the G1/S transition (36), whereas CDK2/
cyclin A and CDK2/cyclin E phosphorylate pRB at the G1 to S transition
(34,37).
When quiescent cells enter the cell cycle owing to mitogenic signals, the
expression of the cyclin Ds is induced and the CDK4/cyclin D and CDK6/
cyclin D complexes are formed as the cells progress through the G1 phase (38).
The kinase activity of the complexes is then activated when they enter the cell
nucleus and are phosphorylated by CAK. This allows the complexes to phos-
phorylate target proteins such as pRBs (39,38). Thus, the cyclin D protein types
play a pivotal role in G1 by transmitting the mitogenic signal to the pRB/E2F
pathway. Notably, cyclin D1 also seems to play a CDK-independent role as a
modulator of transcription factors because it interacts with histone acetylases
and components of the transcriptional machinery. The cyclin D1–deficient
mouse is viable but does have developmental abnormalities that are limited to
restricted tissues (38). Proteasomal degradation of cyclin D1 is triggered by its
phosphorylation on a single threonine residue (Thr-286) by glycogen synthase
kinase-3`(40).
Cyclin E, which regulates CDK2 and possibly also CDK3, is expressed in
late G1 and early S phase (34). The level of cyclin E is abruptly decreased by
proteolysis after polyubiquitination mediated by SCF (Skp2) ubiquitin ligase
(41). Cyclin E regulates the initiation of DNA replication by phosphorylating
components of the DNA replication machinery (39). The CDK2/cyclin E com-
plex also triggers the duplication of centrosomes at G1/S phase by phosphory-
lating the multifunctional protein nucleophosmin (also known as B23) (42,43).
CDK2/cyclin E also targets NPAT (nuclear protein mapped to the AT locus) as
a phosphorylation substrate, which may explain why CDK activity is linked to
the periodic synthesis of histones (44–46).
Cyclin A can activate two different CDKs and functions in both S phase and
mitosis(47). Cyclin A starts to accumulate during S phase and is abruptly de-
stroyed before metaphase. The synthesis of cyclin A is mainly controlled at the
transcription level and involves E2F and other transcription factors. It is still
unknown why CDK2/cyclin A and CDK2/cyclin E complexes are both required
for the initiation of DNA replication and why their order of activation is tightly
regulated. Using a cell-free system, it has been shown that cyclin E stimulates
replication complex assembly by cooperating with Cdc6, a regulator of the
initiation of DNA replication, whereas cyclin A has dual functions: first, it
activates DNA synthesis by the replication complexes that are already as-

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 7
sembled, and second, it inhibits the assembly of new complexes (48). This
regulatory mechanism allows cyclin E to promote replication complex assem-
bly while cyclin A blocks this assembly. Thus, the dual functions of cyclin A
ensure that the assembly phase (G1) ends before DNA synthesis (S) begins,
thereby preventing re-initiation until the next cell cycle.
2.1.2. INK4 Family of CDK Inhibitors
CDK activity is negatively controlled by association with CDK inhibitors
(CKIs), which inactivate CDK/cyclin complexes and thereby cause growth ar-
rest(39,17). CKIs are grouped into either the INK4 (inhibitors of CDK4) fam-
ily or the CIP/KIP family based on their structure and which CDK they target.
There are four INK4 CKIs and three CIP/KIP CKIs (seeSubheading 2.1.3.).
The first class of inhibitors includes the INK4 proteins, which specifically in-
hibit CDK4/cyclin D1-associated kinase activity and are therefore specific for
early G1 phase (49) . Four such proteins have been identified{\}p16
INK4a
,
p15
INK4b
, p18
INK4c
, and p19
INK4d
(39). These INK4 CKIs compete with cyclin
D for binding to CDK4 and consequently cause CDK4/cyclin D complexes to
dissociate. Note that the major portion of these molecules is composed of four
(p16
INK4a
and p15
INK4b
) or five (p18
INK4c
and p19
INK4d
) tandem ankyrin (ANK)
repeats(Fig. 1).This 33-residue repeat was first discovered in ANK, a mem-
brane protein of red cells. Later this motif was found in a wide variety of pro-
teins(50). The beta hairpin helix-loop-helix folds formed by the multiple
tandem ANK repeats stack in a linear manner to produce an elongated struc-
ture that is considered to be involved in macromolecular recognition. p16
INK4a
,
which consists of four ANK repeats (Fig. 1),represents the minimal ANK fold-
ing unit (51).
Of these CKIs, only the p16
INK4a
gene (also known as major tumor suppres-
sor 1 or MTS-1) has been classified as a tumor suppressor by the genetic crite-
ria of loss of heterozygosity (LOH) (49). Unlike other tumor suppressor genes,
p16is often silenced by homozygous deletions at the p16
INK4a
locus (9p21)
that also often inactivate two other important genes nearby, namely p15INK4b
andp14ARF (seeSubheading 2.3.).
2.1.3. CIP/KIP Family of CDK Inhibitors
The second family of CDK inhibitors is composed of the more broadly act-
ing CIP/KIP proteins, such as p21
CIP1/WAF1
, p27
KIP1
, and p57
KIP2
, which inhibit
the activities of cyclin D-, E-, and A-dependent kinases and induce cell cycle
arrest(39). Thus, these CKIs are not specific for a particular phase of the cell
cycle. Unlike the INK4 proteins, the CIP/KIP proteins associate with the CDK-
cyclin complexes and thus do not dissociate these complexes (39,52). The CIP/
KIP proteins share a homologous domain at their N-termini that is believed to

8 Nojima
Fig. 1. The INK4a/ARFlocus encodes p16
INK4a
and p19
ARF
, two degenerated tumor
suppressor proteins. Absence of p16
INK4a
(composed of ANK) activates CDK4(6)/
cyclin D kinase, which phosphorylates pRB, thereby activating the E2F/DP1 tran-
scription factor. Since the transcription of the ARFgene is regulated by E2F, expres-
sion of ARF is induced. ARF separates MDM2 from p53 by recruiting MDM2 into the
nucleolus, possibly in collaboration with cyclin G1 and cyclin G2. This stabilizes p53
in the nucleus and allows it to activate the expression of the target genes that function
in cell cycle checkpoint, DNA repair, and apoptosis. Thus, the INK4a/ARFlocus in-
fluences both the pRB-E2F and p53 pathways.

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 9
participate in the CKI role of these proteins. This amino-terminal domain con-
tains characteristic motifs that are used for binding to both the CDK and cyclin
subunits. This association of CIP/KIP proteins with CDK/cyclin complexes
has an inhibitory effect. For example, it inhibits CDK2 activity by preventing
its Thr-160 phosphorylation by CAK.
p21
WAF1
was discovered almost simultaneously by several studies, each of
which employed different approaches. It was discovered as a CDK2-associ-
ated protein by a two-hybrid system (53), as a senescent cell-derived inhibitor
(Sdi1) of cellular growth (54), as a potential mediator of p53 tumor suppres-
sion that was named wild-type p53-activated fragment (WAF1) (55), and as a
biochemically isolated CDK2/cyclin-binding protein (56). Transcription of the
p21WAF1gene can be induced by the tumor suppressor p53 (55), by the
antimitogenic cytokine transforming growth factor (TGF)-`(57), and by the
phorbol ester tetradecanoyl-phorbol acetate (TPA), which is a protein kinase C
activator(58).
Apart from their role as CDK inhibitors, the CIP/KIP proteins may also act
to bridge the bond between CDK4 (or CDK6) and cyclin D protein types (but
not other CDKs or cyclins), thereby enhancing this association and promoting
the recruitment of the CDK/cyclin D complexes to the nucleus (52,59,60). A
fact supporting this notion is that mouse fibroblasts that lack both p21
WAF1
and
p27
KIP1
are unable to assemble detectable amounts of CDK/cyclin D com-
plexes, and fail to efficiently direct cyclin D proteins into the nucleus (61).
These effects were reversed by returning the CKIs to these cells. Thus, unlike
the INK4 family proteins, the CIP/KIP CKIs promote the formation of CDK4/
cyclin D complexes (62).
In the nucleus, p21
WAF1
binds to proliferating cell nuclear antigen (PCNA)
and blocks DNA replication (63–65) . p21
WAF1
may also regulate the transcrip-
tion of the genes involved in growth arrest, senescence, aging, or apoptosis
after DNA damage (52).
2.1.4. Retinoblastoma Family of Proteins and E2F
The tumor suppressor pRB is the protein product of the retinoblastoma (Rb)
susceptibility gene that is required for the arrest in the G1 phase of the cell
cycle(66,67). pRB, and the related p107 and p130 proteins, share structural
and functional properties and interact with a number of common cellular tar-
gets. Consequently, they form together the “pocket protein family” (36,68)
(Fig. 1). They function as transcriptional repressors in the nucleus and inhibit
the activity of the E2F (early gene 2 factor) transcription factor that regulates
the expression of the many genes required for S phase entry and DNA synthe-
sis(69). Although the pRB proteins do not interact directly with DNA, they
repress E2F-regulated genes in two ways. First, they directly bind to the
transactivation domain of E2F proteins to repress their transcriptional activity.

10 Nojima
Second, they recruit chromatin remodeling enzymes such as histone
deacetylases (HDACs) or methyl transferases to the nearby surrounding
nucleosome structure (46,70).
In the early G1 phase, pRBs are not phosphorylated and can associate with
more than eighty proteins, including the E2F family of transcription factors.
As G1 progresses, however, the pRB proteins become phosphorylated on mul-
tiple serine and threonine residues, primarily by the CDK4/cyclin D or CDK6/
cyclin D complexes, but also partly by CDK2/cyclin E (71). This
hyperphosphorylation inactivates the pRB proteins and causes them to release
their cargo proteins, which activates the cargo proteins and allows them to
mediate the events that are required for further cell cycle progression (39,72).
The phosphorylation of pRB by the CDK4(6)/cyclin D complexes releases hi-
stone deacetylase, which alleviates transcriptional repression, whereas the
phosphorylation by CDK2/cyclin E disrupts the pocket domain of pRB, caus-
ing the pRB–E2F complex to dissociate (73). Recently, it was suggested that
the acetylation of pRB proteins may also influence their activity (74).
The E2F transcription factor was first identified as a transcriptional activity
that influences the adenovirus E2 gene promoter (75). The family of E2F-re-
lated proteins include E2F1–E2F6. These proteins have conserved DNA-bind-
ing and dimerization domains, and three heterodimeric partners, namely DP1,
DP2, and DP3 (76,77). Of the six E2Fs, E2F6 is exceptional in that it lacks the
domains required for transactivation and pRB binding that are normally in the
E2F carboxy-termini. E2F6 appears to play a pRB-independent role in gene
silencing and modulation of G0 phase (78,79). Recent studies have expanded
the roles that are played by the E2F family of transcription factors. It now
appears that apart from being transcriptional regulators of genes involved in
DNA metabolism and DNA synthesis, these proteins also seem to play con-
trasting roles in transcriptional activation and repression, proliferation and
apoptosis, tumor suppression and oncogenesis, and possibly differentiation and
DNA repair (66,69,76,80,81).
2.1.5. Cdc25A Phosphatase
Cdc25, a dual-specificity phosphatase, removes inhibitory phosphates from
the tyrosine and threonine residues of CDKs and thereby promotes cell cycle
progression(82). Three Cdc25 homologs, namely Cdc25A, Cdc25B, and
Cdc25C, have been identified in mammalian cells. Cdc25C promotes the G2/
M transition by activating CDK1 (Cdc2), whereas Cdc25B is proposed to act
as a “starter phosphatase” that initiates the positive feedback loop at the entry
into M phase (83,84) . In contrast, Cdc25A plays an important role in the G1/S
transition(85). Overexpression of Cdc25A activates CDK2/cyclin E or CDK2/
cyclin A by inducing CDK2 tyrosine dephosphorylation. The activated CDK2/

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 11
cyclin complexes then abrogate checkpoint-induced arrest in S phase
(86,104,199). Without Cdc25A activity, the inhibitory tyrosine phosphoryla-
tion of CDK2 would persist, which would maintain the block of entry into S
phase and DNA replication. Cdc25A and Cdc25B (but not Cdc25C) are poten-
tial human oncogenes that have been found to be overexpressed in a subset of
aggressive human cancers (87,88).
To activate the protein kinase activity of CDK2, it must be phosphorylated
on Thr-160 but not on Thr-14 and Tyr-15 (89) . A multi-subunit enzyme CAK,
which consists of cyclin H and CDK7, phosphorylates CDK2 on Thr-160
(90,91), whereas the KAP phosphatase dephosphorylates Thr-160 in the ab-
sence of cyclin, thereby rendering CDK2 inactive (92) . CDK2 is phosphory-
lated on Thr-14 and Tyr-15 by Wee1/Mik1-related protein kinases, whereas
the Tyr-15 residue and possibly also the Thr-14 residue are dephosphorylated
by Cdc25A (93). Thus, downregulation of Cdc25A leads to growth arrest in
late G1 (94). Transcription of the cdc25Agene is inhibited by the E2F-4/p130
complex, which recruits histone deacetylase to the E2F site of the cdc25Apro-
moter in response to TGF-`(95).
As a cellular response to ultraviolet light (UV)-induced DNA damage,
Cdc25A is highly degraded by ubiquitin- and proteasome-dependent proteoly-
sis(96). The same degradation also occurs after hydroxyurea (HU)-triggered
stalling of replication forks (97)as well as during the midblastula transition in
Xenopusembryos under physiological conditions (98). Following DNA dam-
age, Cdc25A is phosphorylated by checkpoint kinase 1 (CHK1) or checkpoint
kinase 2 (CHK2) (Fig. 2B).This phosphorylation is recognized as a tag by the
proteolysis system and Cdc25A is degraded. Thus, CHK1 or CHK2 induces
the G1/S phase checkpoint by phosphorylating Cdc25A (see Subheading 4.2.).
Supporting this is the finding that the elimination of CHK1 expression through
the use of siRNA not only abrogated the S or G2 arrest, it also protected
Cdc25A from degradation(88). During the basal turnover in unperturbed S
phase, CHK1 phosphorylates serines 75, 123, 178, 278, and 292 of Cdc25A. In
contrast, ionizing radiation (IR)-induced Cdc25A proteolysis is mediated by a
combined action of CHK1 and CHK2. Thus, a CHK1-CHK2 inhibitor may be
useful in cancer chemotherapy, as it may potentiate the cytotoxicity caused not
only by DNA-damaging drugs that induce G2 arrest but also by agents that
promote S arrest (99,100).
2.1.6.Myc
The c-mycprotooncogene is a pivotal regulator of cellular proliferation,
growth, differentiation, and apoptosis (101). The Myc family proteins (Myc,
N-Myc, and L-Myc) are transcription factors with basic helix-loop-helix leu-
cine zipper protein (bHLH-ZIP) motifs that bind to the DNA sequence

12 Nojima
Fig. 2. ATM/ATR and CHK1/CHK2 kinases mediate the signaling network of the
DNA damage and DNA replication checkpoints. (A)Phosphorylation target proteins
of ATM and ATR kinases. There are two parallel pathways that respond to DNA dam-
aging stress in mammalian cells. The ATM pathway responds to the presence of DSBs
acting at all phases of the cell cycle. The ATR pathway not only responds to DSBs but
also to the agents that disturb the function of replication forks. Following their activa-
tion by DSBs or replication stress, ATM/ATR kinases phosphorylate unique (red and
black, respectively) or overlapping (green) target proteins at specific serine (S) or
threonine (T) residues of indicated (if known) numbers. (B)Phosphorylation target

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 13
CACGTG (E-box) when dimerized with Max, another bHLH-ZIP. A head-to-
tail pair of Myc-Max dimers form a heterotetramer that is capable of bridging
distant E-boxes. Mitogen exposure promptly induces the expression of c-myc.
Ectopic expression of c-mycalso encourages quiescent cells to enter into S
phase(102). Myc not only targets genes that encode cyclins D2, D1 and E, and
Cdc25A as a transcription factor, but also sequesters p27
KIP1
into CDK4(6)/
cyclin D complexes away CDK2/cyclin E to cause phosphorylation and subse-
quent ubiquitination and proteasome-mediated degradation of the p27
KIP1
,
thereby realizing at least three distinct regulatory functions of CDK2/Cyclin E
activity, E2F-dependent transcription, and cell growth (103).
In association with Max, Myc binds to the E-boxes in a variety of gene
promoters and thus orchestrates the transcriptional activation of a diverse set
of genes. However, Myc on its own inhibits the transcription of other genes,
includingp21WAF1(104)and another cyclin-dependent kinase inhibitor,
p15INK4b(105,106). The DNA-binding protein Miz-1 directly recruits Myc
to the p21WAF1promoter, where Myc selectively inhibits bound p53 from
activatingp21WAF1transcription and favors the initiation of apoptosis (107).
Thus, Myc can influence the outcome of a p53 response in favor of cell death.
2.2. The p53-pRB Pathway Controls the G1/S Transition
Thep53tumor suppressor gene (TP53) is the most frequently mutated gene
(about 50%) in human tumors, and encodes a 53 kDa transcription factor (p53)
that directly induces the expression of a substantial number of genes that are
important for cell cycle regulation, DNA damage repair, and apoptosis
(108,109). Of the genes that are induced by p53, p21
WAF1
plays a pivotal role
in G1 arrest by inhibiting CDK4(6)/cyclin D1 activity, thereby reducing the
phosphorylation of pRB and promoting G1 arrest of the cell cycle. This inter-
connecting signaling pathway involving p53, pRB, and E2F plays an essential
role in G1/S transition of the cell cycle. p21
WAF1
is also known to inhibit S
phase progression (G1 arrest) by binding to PCNA, a ring protein that pro-
motes DNA replication (63). The expression level of p53 is low in the absence
Fig. 2. (continued)proteins of CHK1 and CHK2 kinases. CHK2 is primarily phospho-
rylated by ATM (and partially by ATR), whereas CHK1 is phosphorylated by ATR.
Then, CHK1/CHK2 kinases transmit the checkpoint signals by phosphorylating unique
(red and black, respectively) or overlapping (green) target proteins at specific serine
(S) or threonine (T) residues of indicated numbers. CHK2, phosphorylated on Thr-68
by ATM, is activated to autophosphorylate on Thr-383 and Thr-387 (blue arrows),
further enhancing its kinase activity. Ser-46 of p53 is presumed to be phosphorylated
by putative p53 S46 kinase. These phosphorylated proteins further propagate the sig-
nal to the downstream targets, thereby regulating various cellular events.

14 Nojima
of cellular stress. However, various types of stress, including DNA damage,
induce p53 expression and cause G1 arrest. In cases where the DNA damage is
too severe to be repaired, p53 induces apoptosis as a desperate attempt to pro-
tect the organism (19,110, 111). This essential role of p53 as a critical brake on
tumor development explains why it is so frequently found in cancer cells
(112,113).
Other genes that are upregulated by p53 (112)includecyclin G1(27),MDM2
(murine double murine 2), BAX (bcl2-associated X protein), GADD45(114),
14-3-3m(115),CDK4(116),p53R2(117,118),p53AIP1(119),p53DINP1
(120), and p53RDL1(121). Cyclin G1 and MDM2 regulate the stability of the
p53 protein (seeSubheading 2.3.). Bax forms a homodimer or heterodimer
with Bcl2, and increasing amounts of the Bax homodimer trigger cytochrome-
crelease from mitochondria, thus promoting apoptosis (122). Gadd45 (induced
after growth arrest and DNA damage) is involved in regulating nucleotide ex-
cision repair of UV-damage together with p53 and another p53-downstream
gene,p48XPE(123). The 14-3-3m protein associates with and recruits Cdc25C
from the nucleus to inhibit the activation of CDK1/cyclin B, thus causing G2
arrest. p53R2 is a homolog of ribonucleotide reductase small subunit (R2).
Expression of p53R2, but not that of R2, is induced by DNA damage and serves
to supply the cell with the deoxyribonucleotides needed for DNA repair.
p53RDL1 (p53-regulated receptor for death and life) interacts with its ligand
Netrin-1 and promotes the survival of damaged cells against apoptosis.
At least some of the eleven phosphorylation sites identified on p53 seem to
play pivotal roles in its regulation. Three functionally important domains have
been identified in the p53 molecule, and phosphorylation at these sites is con-
sidered to influence the structural changes of these domains. The middle do-
main constitutes the core domain that associates with the specific nucletotide
sequences at the promoter regions of its target genes. This domain harbors the
vast majority of the p53 “hot spot” mutations found in human cancers. In cells
with damaged DNA, Ser-15 of p53 is phosphorylated by ATM (ataxia telang-
iectasia mutated) or ATR (ATM-Rad3-related) (124–126). The ATM gene was
first isolated from patients with the autosomal recessive disorder ataxia telang-
iectasia (A-T). These patients exhibit cerebellar degeneration, immunodefi-
ciency, radiation sensitivity, and predisposition to cancer (127).
Phosphorylation of p53 at Ser-20 by CHK1 or CHK2 may also be important
for regulating the interaction between p53 and MDM2 (128,129) . Upon severe
DNA damage, Ser-46 on p53 is phosphorylated and apoptosis is induced. As
p53AIP1 (p53-regulated apoptosis-inducing protein 1) is selectively induced
by p53 molecules that have been phosphorylated at Ser-46, it may be that
p53AIP1 mediates this p53-dependent apoptosis by inducing the release of
cytochrome-cfrom mitochondria (130) . p53DINP1 (p53-dependent damage-

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 15
inducible nuclear protein 1) functions as a cofactor of the putative p53-Ser46
kinase that promotes phosphorylation of p53 at Ser-46 (120).
2.3. Regulation of p53 Stability by ARF and MDM2
TheINK4alocus that generates p16
INK4a
also encodes a degenerated gene
product called ARF (after alternative reading frame) (131). Thus, the locus
encodes two tumor suppressor proteins, p16
INK4a
and p19
ARF
(p14
ARF
in
humans), which activate the growth suppressive functions of pRB and p53,
respectively(67). ARF is a highly basic (pI > 12), arginine-rich nucleolar pro-
tein(132). Deletion of the ARFgene can inactivate p53 function in tumors
where p53 itself remains intact. Transcription of the ARFgene is regulated by
E2F, and thus the INK4a/ARFlocus influences both the pRB-E2F and p53
pathways.
Overexpression in the same tumor lines of MDM2 (murine double murine
2; Hdm2 in humans), a protein whose expression is upregulated by p53 (see
Subheading 2.2.), has the same effect. This is because ARF binds to MDM2
and abrogates its p53-inhibitory activity. MDM2 destabilizes p53 by catalyzing
its ubiquitination by acting as an E3 ubiquitin ligase. This promotes the nuclear
export of p53, thereby allowing it to be targeted for proteasomal degradation
(133,134). Actually, MDM2 is frequently overexpressed in human tumors, and
this leads to the rapid degradation of p53 (135). Since MDM2 directly binds to
the N-terminus of p53, phosphorylations of p53 at Ser-15, Ser-20, and Thr-18
are important for the dissociation of MDM2 from p53 (128,129). MDM2 is
itself transcriptionally activated by p53, which thus creates a negative feed-
back loop. Consequently, inhibiting the interaction between p53 and MDM2
by the application of synthetic molecules may serve as an effective cancer treat-
ment because it may lead to cell cycle arrest or apoptosis in p53-positive tumor
cells(109).
Other proteins also modulate MDM2 activity. Mitogen-induced activation
of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, the
AKT/PKB serine-threonine kinase, results in the phosphorylation of MDM2
on Ser-166 and Ser-186. CDK2/cyclin A also phosphorylates MDM2 on Thr-
216(136). These phosphorylation events are necessary for the translocation of
MDM2 from the cytoplasm into the nucleus and thus serve to promote the p53-
inhibitory activity of MDM2 as a ubiquitin ligase (134). Cyclin G1 directly
binds to MDM2 (137), recruits PP2A (protein phosphatase 2A) to dephospho-
rylate MDM2 at Thr-216, and releases MDM2 from p53, thereby cooperating
with ARF to restrict the ability of MDM2 to negatively regulate p53 (138)
(Fig. 1).Indeed, cyclin G1
-/-
mouse embryo fibroblasts show enhanced accu-
mulation of p53 and are partially deficient in an irradiation-induced G2/M-
phase checkpoint (139) . Cyclin G2 may also have redundant or compensatory

16 Nojima
functions, because it associates with many of the same proteins to which cyclin
G1 binds, including p53, PP2A, MDM2, and ARF (137). This p53-stabilizing
effect of PP2A/cyclin G complexes may also influence the malignancy of can-
cer cells, considering that enhanced expression of a truncated form of PP2A
was observed in highly metastatic melanoma cells (140). Cells overexpressed
with this truncated form of PP2A show irradiation-induced checkpoint defects
and appear to elevate genetic instability, which may promote tumor progres-
sion(141). These data suggest that cyclin G1 is a positive feedback regulator
of p53, since it downregulates the activity of MDM2, which would otherwise
restrain the accumulation of p53 (142) .
3. DNA Damage Checkpoints
DNA damage caused by IR, chemical reagents, or similar environmental
insults induces cell cycle arrest at G1, S, or G2, thereby preventing the replica-
tion of damaged DNA or aberrant mitosis until the damage is properly repaired.
The molecular mechanism in mammalian cells that detects the presence of
double-strand breaks (DSBs) is not well understood. Research in the budding
yeastSaccharomyces cerevisiae,however, tells us that a quintet complex com-
posed of RAD24, RFC2, RFC3, RFC4, and RFC5 acts in this organism as a
sensor of DSBs (143) . Disruption of these components causes defects in the
damage checkpoint machinery of S. cerevisiae (144). The same DSB-sensing
mechanism is also used in another useful yeast strain, Schizosaccharomyces
pombe(145,146). In S. cerevisiae,the signal of the DSB abnormality is trans-
mitted to the ring-shaped hetero-trimer that is composed of Ddc1/Rad17/Mec3
(Rad9/Rad1/Hus1 in fission yeast and mammals). This hetero-trimer resembles
the replication factor PCNA (147). In fission yeast, this complex activates Rad3
kinase, which then phosphorylates CHK1 (148). The activated CHK1 then tar-
gets Cdc25C for phosphorylation. Cdc25C is subsequently recognized by
Rad24, a 14-3-3 protein (seeSubheading 2.2.), which recruits it out from the
nucleus into the cytoplasm, where it inactivates the CDK1/cyclin B complex
(Cdc2/Cdc13 in S. pombe), which results in G2/M arrest (149). The 14-3-3
proteins bind to serine/threonine-phosphorylated residues in a specific manner
and regulate key proteins involved in various physiological processes such as
the cell cycle, intracellular signaling, apoptosis, and transcription regulation
(150). Similar checkpoint regulatory mechanisms involving 14-3-3 proteins
are also employed in vertebrate cells (2,86,151). For example, human CHK1 is
activated by phosphorylation and thereby phosphorylates Cdc25C on Ser-216,
which is recognized by the 14-3-3mprotein. The14-3-3m protein then removes
Cdc25C from the nucleus to the cytoplasm, thereby preventing the activation
of the CDK1/cyclin B complex and entry into mitosis (3,152–154).

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 17
In mammalian cells, there are two parallel pathways that respond to DNA-
damaging stresses (Fig. 2).The first pathway is the ATM pathway, which re-
sponds to the presence of DSBs at all phases of the cell cycle. The second
pathway is the ATR (ATM-Rad3-related) pathway, which responds not only to
DSBs but also to the agents that interfere with the function of replication forks
(126,4,127). A third pathway may involve the newly identified ATX (ATM-
related X protein), which phosphorylates and activates CHK1 and/or CHK2
(126,127,155). As shown in Fig. 2,ATM phosphorylates many target proteins
at their specific serine or threonine residues and activates their functions. In
response to IR, for example, ATM phosphorylates RAD9 on Ser-272 (156);
PLK3, which further phosphorylates CHK2, contributing to its full activation
(157); SMC1 (the cohesin protein) on Ser-957 and Ser-966 (158,159); H2AX
on Ser-140, which is required for 53BP1 accumulation at DNA break areas
(160); 53BP1 on Ser-6, Ser-25/Ser-29, and Ser-784 (161); and MDC1 (162).
The human ATR protein complexes stably with a protein called ATRIP
(ATR-interacting protein). These complexes localize in nuclear foci after dam-
age and thus appear to be recruited to the sites of DNA damage (163). The
ATR homologs in fission yeast (Rad3) and budding yeast (Mec1) also form
similar complexes with the ATRIP-related factors Rad26 and Ddc2/Lcd2/Pie1,
respectively, which are also recruited to the sites of DNA damage (164–167).
ATR phosphorylates H2AX on Ser-139 (168), whereas ATM/ATR phospho-
rylate E2F on Ser-31 (169) . The checkpoint functions of ATM in response to
IR are primarily mediated by the effector kinase CHK2, whereas those of ATR
in response to replication inhibition and UV-induced damage are mediated by
CHK1. Thus, the structurally unrelated CHK2 and CHK1 proteins channel the
DNA damage signals from ATM and ATR, respectively (21,170). However,
recent observations suggest the existence of various “crosstalks” among these
kinases(100,171), and the presence of a novel checkpoint cascade signaling by
way of ATM-CHK1 to Tousled-like kinases (TLKs) that causes chromatin re-
modeling in response to various stresses (172).
The expression of the labile CHK1 protein is restricted to the S and G2
phases(173). Although it is active even in unperturbed cell cycles, it is further
activated in response to DNA damage or stalled replication (100,174). Follow-
ing a checkpoint signal, CHK1 is phosphorylated on Ser-317 and Ser-345 by
ATR in cooperation with the sensor complexes, which include the mammalian
homologs of Rad17 and Hus1. The phosphorylation at the Ser-345 site is
required for nuclear retention of CHK1 following an HU-induced checkpoint
signal(175–177). CHK1 not only stimulates the kinase activity of DNA-
dependent protein kinase (DNA-PK) complexes, which leads to increased
phosphorylation of p53 on Ser-15 and Ser-37; it also elevates the DNA-PK-

18 Nojima
dependent end-joining reactions, thereby promoting the repair of DSBs (178).
CHK1
–/–
mice show a severe proliferation defect and death in embryonic stem
(ES) cells and peri-implantation embryonic lethality. The ES cells lacking
CHK1 have also been shown to have a defective G2/M DNA damage check-
point in response to IR (179,180). In contrast, CHK1-deficient cells called
DT40 are viable, but they fail to arrest at G2/M in response to IR and fail to
maintain viable replication forks when DNA polymerase is inhibited (181).
In contrast, the other Ser/Thr protein CHK2 kinase (also known as hCds1)
must be phosphorylated at Thr-68 by ATM to activate it in response to IR-
induced DNA damage (this is not the case for damage owing to UV or HU)
(170,182). Unlike CHK1, CHK2 is a stable protein that is expressed through-
out the cell cycle and that seems to be inactive in the absence of DNA damage
(173). Its activation involves its dimerization and autophosphorylation. Unlike
the catalytically inactive form of CHK2, wild-type CHK2 leads to G1 arrest
after DNA damage by phosphorylating p53 on Ser-20, which causes the pre-
formed p53/MDM2 complexes to dissociate and increases the stability of p53
(128). Unlike ATM
–/–
andp53
–/–
mice,CHK2
–/–
mice do not spontaneously
develop tumors, although the IR-induced G1/S cell cycle checkpoint—but not
the G2/M or S phase checkpoints—was impaired in primary mouse embryonic
fibroblasts (MEFs) derived from CHK2
–/–
mice (183,184).
That the fission yeast homolog of CHK2, Cds1, may participate in repair is
suggested by the finding that it interacts with the Mus81–Eme1 endonuclease
complex, which can resolve the Holliday junction (185,186). The human
Mus81–Eme1 complex also has a similar function as a flap/fork endonuclease
that is likely to play a role in the processing of stalled replication fork interme-
diates(187).
CHK1 and CHK2 share partly redundant roles in that they target common
downstream effector proteins such as the Polo-like kinase 3 (PLK3) (188), the
promyelocytic leukemia (PML) protein (189), the E2F1 transcription factor
(190), or the TLKs (172). PLK3 binds to and phosphorylates p53 on Ser-20.
Through this direct regulation of p53 activity, PLK3 is at least partly involved
in regulating the DNA damage checkpoint as well as M-phase function. The
PMLgene is translocated in most acute promyelocytic leukemias and encodes
a tumor suppressor protein that plays a pivotal role in gamma irradiation–in-
duced apoptosis. It is proposed that CHK2 mediates gamma irradiation–in-
duced apoptosis in a p53-independent manner through an ATM-CHK2-PML
pathway(189). PML also recruits CHK2 and p53 into the PML-nuclear bodies,
and enhances the p53/CHK2 interaction to protect p53 from MDM2-mediated
ubiquitination and degradation (191). Mutations in the prototypic member of
the Tousled (Tsl) kinase from the plant Arabidopsis thaliana lead to a pleiotro-
pic phenotype (192) . In mammals, however, the TLKs are regulated in a cell

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 19
cycle-dependent manner that peaks at S phase and are involved in chromatin
assembly by phosphorylating the chromatin assembly factors Asf1a and Asf1b
(193). CHK1 phosphorylates TLK1 on Ser-695 in vitro, and substitution of
Ser-695 with alanine impairs the efficient downregulation of TLK1 after DNA
damage(172).
4. G1 Checkpoint Response
In mid-to-late G1, and if the cellular environment is favorable for prolifera-
tion, a binary decision—whether to commit to the mitotic cell cycle and enter
S-phase, or whether to not commit to the cell cycle and remain in a quiescent,
non-proliferative state—is made at the “restriction point” (194,195). As de-
scribed above, many proteins are involved in making this critical decision and
in ensuring proper progression of the G1/S transition. Although cyclin D meets
the criteria of the critical restriction point factor, the system seems to be far
more complex than just relying on a single factor. Moreover, the relationship
between the restriction point and DNA damage checkpoints remains elusive
(196). The cell cycle checkpoints that monitor the proper G1/S transition and S
phase progression during potentially hazardous genotoxic stress (103, 197)will
be discussed in the following sections.
4.1. The ATM(ATR)/p53-Mediated Pathway
The ATM(ATR)/p53 pathway plays a pivotal role in one of the checkpoint
mechanisms that arrest the cell cycle at G1 phase following DNA damage (G1
checkpoint)(Fig. 3).As described in Subheading 3, ATM is activated in re-
sponse to IR, whereas ATR is activated in response to replication inhibition or
UV-induced damage. The activated ATM or ATR then phosphorylates p53 (on
Ser-15), and this phosphorylation causes MDM2 to dissociate from p53, which
stabilizes p53 and leads to its accumulation (128,129). Increased expression of
ARF owing to E2F1 stabilization in response to DNA damage also blocks the
inhibitory function of MDM2, thereby increasing the nuclear amount of p53.
The principal kinases relaying ATM(ATR)-initiated checkpoint signaling
are preferentially CHK2 for ATM and CHK1 for ATR. In response to IR or
DNA replication stress, ATR phosphorylates CHK1 at Ser-317 and Ser-345
(175,177,198), which moderately increases its kinase activity and allows it to
propagate the signal to downstream effectors, including p53, which CHK1
phosphorylates on Ser-20 (129). In response to IR, ATM phosphorylates CHK2
at Thr-68 (199), followed by CHK2 autophosphorylation on Thr-383 and Thr-
387 and the activation of several target proteins, including p53, which CHK2
also phosphorylates on Ser-20 (128,129) . These Ser-20 phosphorylation events
both induce MDM2 to dissociate from p53.

20 Nojima
Fig. 3. ATM(ATR)-mediated G1/S checkpoint pathways. DNA damage triggers a
rapid cascade of phosphorylation events involving either the ATM and CHK2 (upon
IR) or the ATR and CHK1 (upon UV light) kinases. These phosphorylation events
activate the target protein kinases to trap and phosphorylate the next target proteins,
thereby transmitting the DNA damage signals. It has been determined that in response
to IR, ATM phosphorylates CHK2 at Thr-68, whereas ATR (or ATM) phosphorylates
CHK1 at Ser-317 and Ser-345. In one pathway (left), the CHK2 or CHK1 kinase phos-
phorylates Cdc25A phosphatase at serines 75, 123, 178, 278, and 292 (100). Of these,
the Ser-123 residue that is targeted by CHK2 and the Ser-75 residue that is phosphory-
lated by CHK1 seem to be particularly critical residues of Cdc25A (202, 99). The
phosphorylated Ser-123 or Ser-75 residue is recognized by the ubiquitination (Ub)
enzyme, and this promotes the rapid degradation of Cdc25A by the proteasome. Due
to the disappearance of Cdc25A phosphatase activity that this degradation causes, the
CDK2/cyclin E complex is locked in its inactive form because of the presence of the
inhibitory phosphorylation on the Thr-14 and Tyr-15 residues of CDK2. Thus, the
CDK2/cyclin E complex fails to load Cdc45 onto chromatin and the blockade of the
initiation of the DNA replication origins is maintained.

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 21
The stabilized and activated p53 protein that results from CHK1/CHK2-
mediated phosphorylation induces the transcription of a large number of genes,
includingp21
WAF1
, which silences the kinase activities of the CDK2/cyclin E,
CDK2/cyclin A, or CDK4(6)/cyclin D complexes. This prevents the complexes
from loading the Cdc45 origin binding factor onto chromatin, which precludes
the recruitment of DNA polymerases, thereby blocking initiation of DNA rep-
lication from the unfired origins (200,201). Another important consequence of
inhibiting both the CDK2 and CDK4(6) kinase complexes is that these com-
plexes cannot then phosphorylate pRB, which allows pRB to maintain its inhi-
bition of the E2F-dependent transcription of S-phase genes that are essential
for S-phase entry as described in Subheading 2.1.4.These effects all result in
G1 arrest. Maintenance of the G1/S arrest by way of this pathway after DNA
damage is a delayed response that requires the transcription, translation, and/or
protein stabilization of key checkpoint transducers. However, once initiated,
this pathway provides a long-lasting G1 arrest, and the entry into S phase is
prevented as long as a single unrepaired DNA lesion is detected by the check-
point machinery.
4.2. The ATR(ATM)/Cdc25A-Mediated Pathway
The human Cdc25A phosphatase plays a pivotal role at the G1/S transition
because it enhances the kinase activities of the CDK2/cyclin E and CDK2/
cyclin A complexes by dephosphorylating the inhibitory phosphorylated Thr-
14 and Tyr-15 residues of CDK2 (102197). After UV and IR exposure, Cdc25A
is ubiquinated because it is phosphorylated by CHK1 (in the case of UV)
Fig. 3. (continued)In the other pathway (right), Ser-15 of p53 is directly phospho-
rylated by ATM or ATR in cells with damaged DNA. The phosphorylation of p53 at
Ser-20 by CHK1 or CHK2 induces the dissociation of the p53/MDM2 complex, which
increases the stability of p53 because MDM2 primes p53 for ubiquitination and
proteasomal degradation (seeFig. 1). CHK1 also stimulates the kinase activity of
DNA-PK complexes, which increases the phosphorylation of p53 on Ser-15 and Ser-
37. Furthermore, DNA damage can also upregulate ARF, which specifically inhibits
MDM2, putatively in collaboration with the cyclin G proteins. The collective result is
that stable and transcriptionally active p53 transcription factor accumulates in the cell
nucleus and induces the expression of a large number of target genes, including the
p21 CDK inhibitor. The increased p21 levels inhibit the CDK2/cyclin E complex or
the CDK4(6)/cyclin D complexes, thus arresting the cell cycle at G1/S phase. Upon
severe DNA damage, Ser-46 on p53 is phosphorylated by the putative p53-Ser46 ki-
nase with the aid of p53DINP1, which selectively induces the expression of p53AIP1,
which is a mediator of apoptosis because it induces the release of cytochrome-c from
mitochondria.

22 Nojima
(87,96,)and CHK2 (in the case of IR) (202). The critical residue of Cdc25A
that is targeted by CHK2 is Ser-123 (202). Cdc25A is also phosphorylated on
Ser-75 by CHK1 (109). The phosphorylated Ser-123 residue (and possibly also
the phosphorylated Ser-75 residue) is recognized by the ubiquitination (Ub)
enzyme, which promotes the rapid degradation of Cdc25A by the proteasome
system. Removal of Cdc25A in turn keeps the CDK2-associated kinase com-
plexes in their inactive form due to the persisting inhibitory phosphorylation of
their Thr-14 and Tyr-15 residues. This results in G1 arrest. The important tar-
get of this cascade is the inhibition of CDK2-dependent loading of Cdc45 onto
DNA pre-replication complexes. Thus, the ATM(ATR)–CHK2(CHK1)–
Cdc25A–CDK2 pathway accounts for the rapid and p53-independent initiation
of the G1 checkpoint, where the abundance and activity of Cdc25A decreases
without delay in response to IR- or UV-mediated DNA damage (Fig. 3) . It is
likely that this regulatory mechanism is conserved among vertebrates and op-
erates in every cell type.
During interphase, CDK2 appears to phosphorylate Cdc25A, which consti-
tutes a Cdc25A-CDK2 autoamplification feedback loop (203). Cdc25A also
seems to be involved in the G2/M transition, besides its commonly accepted
effect on G1/S progression (87). Proteolysis of Cdc25A is also linked with the
intra-S-phase checkpoint, which guards against premature entry into mitosis in
the presence of stalled replication forks.
4.3. Other Potential G1 Checkpoint Pathways
It has been reported that there is another G1 checkpoint induced by IR expo-
sure, which is characterized by enhanced protein degradation (204). In this
checkpoint, DNA damage unmasks a cryptic “destruction box” (RxxL) within
the cyclin D1 amino-terminus that is then recognized by the anaphase-promot-
ing complex (APC) ubiquitin ligase, which primes cyclin D1 for rapid
proteasomal destruction (197). This causes the p21
WAF1
protein, which served
as an assembly factor of the CDK4(6)/cyclin D1 complexes, to be released.
p21
WAF1
is then free to bind to another of its targets, the CDK2/cyclin E com-
plex. This binding inactivates the kinase activity of the complex (Fig. 3) . Since
the proliferation of many mammalian somatic cells depends on the presence of
abundant CDK4(6)/cyclin D1 complexes, the destruction of cyclin D1 together
with the inactivation of the S-phase-promoting CDK2/cyclin E strongly in-
duces G1 arrest.
Exposure of epithelial cells to UV light can also lead to yet another G1
checkpoint mechanism. This mechanism involves the gradual accumulation of
p16
INK4a
, which selectively disrupts the CDK4(6)/cyclin D1 complexes. This
again causes the release of p21
WAF1
, which can then bind to and inhibit CDK2/
cyclin E, thereby resulting in G1 arrest. If these mechanisms are confirmed as

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 23
cell cycle checkpoints, they would each serve as examples of an ATM-inde-
pendent, cell-type-restricted response. Note that because cyclins D2 and D3
are not degraded upon DNA damage, these pathways would have little effect
in cell types that express several D-type cyclins or lack cyclin D1.
To ensure the exact duplication of the genome during every cell division,
which is a basic requirement of every proliferating cell, eukaryotes adopt a
strategy that temporally separates the assembly of the pre-replication complex
(pre-RC) from the initiation of DNA synthesis (Fig. 4) (201,205). A key com-
ponent of the pre-RC is the hexameric minichromosome maintenance (MCM)
protein complex, which consists of the six Mcm2–Mcm7 proteins (206). The
MCM complex is presumed to be a helicase functioning in the growing forks,
and like other helicase proteins (207), it actually adopts a toroidal structure
when observed under a microscope (208) . The MCM complex is recruited to
the replication origins, where the two protein kinase complexes Cdc7–Dbf4 (in
budding yeast) and CDK2/cyclin E trigger a chain reaction that results in the
phosphorylation and activation of the MCM complex and finally in the initia-
tion of DNA synthesis (201,209). At the onset of S phase, S-phase kinases
promote the association of Cdc45 with MCM at the origins. Upon the forma-
tion of the MCM–Cdc45 complex at the origins, the duplex DNA is unwound
and various replication proteins, including DNA polymerases, are recruited
onto the unwound DNA (200). A “licensing checkpoint” that prevents passage
into S phase in the absence of sufficient origin licensing may also exist in mam-
malian cells (210).
5. The S-Phase Checkpoint
Proliferating cells are always exposed to life-threatening insults that disturb
the proper replication and segregation of their genomes into daughter cells. In
response to these genotoxic insults, eukaryotic cells have evolved checkpoint
mechanisms that monitor the progression of DNA replication at S phase and
halt replication if an abnormality is observed (Fig. 4) . At least two distinct S-
phase checkpoints seem to exist. One of these occurs in response to DNA-
replication stress that interferes with the proper progression of the replication
forks. The other is an intra-S-phase checkpoint that functions in response to
DSBs(2,4). The S-phase checkpoint may also have a function during an unper-
turbed S phase, because even in the absence of exogenous agents, mutants of
the many genes that are involved in this checkpoint show aberrant checkpoint
signaling, and some mutants also cause checkpoint induction (2).
5.1. S-Phase Checkpoint in Response to DNA Replication Stress
Several types of agents are known to interfere with the function of replica-
tion forks and to elicit the S-phase checkpoint. These include agents that

24 Nojima
Fig. 4. Molecular mechanism of S-phase progression and the S-phase checkpoints.
Upon initiation of DNA replication, DNA replication initiation factors such as
MCM10, CDC45–Sld3 (budding yeast) and RPA and checkpoint complexes bind to
the pre-replicative complex (pre-RC) on chromatin and trigger the unwinding of DNA.
The hexameric MCM2-7 complex is recruited to the replication origins by a number of
proteins, including MCM10, RPA, and CDC45–Sld3. The MCM complex is a puta-
tive helicase of the growing forks. Two protein kinase complexes, Cdc7/Dbf4 (bud-
ding yeast) and CDK2/cyclin E, trigger a chain reaction that results in the
phosphorylation of the Mcm complex and finally the initiation of DNA synthesis. The

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 25
directly inhibit DNA synthesis. For example, HU stalls replication forks by
depleting the deoxynucleotide triphosphate (dNTP) pool, while aphidicolin ac-
tivates the checkpoint by inhibiting DNA synthesis by blocking the activities
of polymerases (Fig. 4) . In addition, DNA-modifying agents that block repli-
cation can elicit the S-phase checkpoint. These agents include methyl
methanesulfonate (MMS) and UV-induced DNA lesions, which slow down
the rate of DNA-replication-fork progression in budding yeast (211).
The study of the DNA-replication checkpoint is most advanced in yeasts.
However, the checkpoint mechanisms that were unveiled in yeasts also seem
to be conserved in mammalian cells (2,149). The central checkpoint kinases
Mec1 (ATR in humans) and Rad53 (CHK2 in humans) play an essential role in
maintaining DNA replication fork stability in response to DNA damage and
replication fork blockage, and they inhibit the activation of late-firing replica-
tion origins after HU and MMS exposure (4). The DNA replication forks ap-
pear to function both as the activator and as the primary effector of the S-phase
checkpoint pathway, since the recruitment of Ddc2 (ATRIP in humans) to
nuclear foci and the subsequent activation of the Rad53 kinase occurs only
during S phase and requires the assembly of the replication forks (212).
In budding yeast, proteins that are essential for DNA replication, such as
DNA polymerase ¡and its interacting partners Dpb11 and Drc1/Sld2, are also
required for efficient checkpoint activation (213) . Dpb11 and its human ho-
molog TopBP1 associate with the PCNA-like protein Ddc1 and human Rad9,
Fig. 4.(continued)ATR/ATRIP (Rad3/Rad26) complex and the Pol_ –primase com-
plex and several other replication proteins are also recruited to the unwound DNA.
After this, the RAD1/RAD9/HUS1 complex binds to chromatin, an event that requires
the RAD17/RFC2-5 complex.
Two kinds of S-phase checkpoint mechanisms are known. One monitors the stalled
replication forks (DNA-replication checkpoint) while the other monitors the replica-
tion block induced by DSBs during S phase (intra-S-phase checkpoint). In contrast to
the checkpoints at the G1/S and G2/M transitions that arrest the cell cycle, these S
phase checkpoints can only delay the progression of S phase. Proteins involved in the
regulation of DNA replication such as DNA polymerase ¡, Dpb11 (TopBP1 in hu-
mans), Drc1/Sld2 (budding yeast), Ddc1 (budding yeast), and RPA are also required
for the S-phase checkpoint in response to replication blockage. Claspin (Mrc1 in yeast)
that is phosphorylated at Ser-864 and Ser-895 by CHK1 also regulates the S-phase
checkpoint. ATM is the master transducer of the S-phase checkpoint and phosphory-
lates BRCA1 and BRCA2 as well as NBS1 (at Ser-343), which is a component of the
NBS1/MRE11/RAD50 complex. CHK1 also phosphorylates TLK (at Ser-695), a pro-
tein kinase that is potentially involved in regulation of chromatin assembly. Acetyla-
tion of nucleosomal histone H3 or H4, which regulates the chromatin structure, and
gene expression also play a role in the S-phase checkpoint.

26 Nojima
respectively, and seem to collaborate in monitoring the progression of replica-
tion forks (214,215) . The Pol_–primase complex and RPA (replication protein
A) are also required for the S-phase checkpoint in response to replication blocks
(216).
Claspin, a CHK1-interacting protein, is required for the ATR-dependent
activation of CHK1 in Xenopusegg extracts that contain incompletely repli-
cated DNA (217) . Claspin, ATR, and Rad17 bind to chromatin independently
and appear to collaborate in checkpoint regulation by detecting different as-
pects of the DNA replication fork (218).Xenopus Claspin may be phosphory-
lated at Ser-864 and Ser-895 by CHK1 (219). Human Claspin is a cell
cycle-regulated nuclear protein whose levels peak at S/G2 phase and that is
phosphorylated in response to replication stress or other types of DNA dam-
age. It appears to work as an adaptor molecule that brings the ATR/CHK1 and
RAD9/RAD1/HUS1 complexes together to regulate the S-phase checkpoint
(220). These observations suggest that the activation of CHK1 by ATR may be
regulated by Claspin in a similar way in budding yeast: Rad9 is phosphory-
lated by Mec1 in response to DNA damage and subsequently serves as a scaf-
fold protein for Rad53, thus allowing Rad53 to autophosphorylate and
self-activate(221). Mrc1, a yeast homolog of Claspin, is also important for the
activation of Rad53 and Cds1 in response to HU, and thus may mediate the
checkpoint response to replication blockage in a similar manner to Claspin
(222,223).
In budding yeast, the S-phase checkpoint activates the ATM-like Mec1 and
the CHK2-related Rad53 kinases in response to stalled replication forks that
arise owing to replication stress or DNA damage in S phase. These kinases in
turn inhibit spindle elongation and late origin firing, which stabilize the DNA
polymerases at the arrested forks (4). Orc 2 (origin recognition complex 2)
plays a pivotal role in maintaining the number of functional replication forks,
and the amount of DNA damage required for Rad53 activation is higher in S
phase than in G2 (224) . For the S-phase checkpoint, acetylation of the nucleo-
somal histone H3 or H4 that regulates chromatin structure and gene expression
also appears to be important (225). Studies in fission yeast suggest that the
signal activating the S-phase checkpoint is generated only when replication
forks encounter DNA damage (226).
5.2. S-Phase Checkpoint in Response to DSBs
After DNA damage, proliferating cells actively slow down their DNA repli-
cation by activating a checkpoint. This gives the cell time to repair the damage.
This checkpoint is often called the intra-S-phase checkpoint (Fig. 4) (4,21).
The intra-S-phase checkpoint consists of regulatory networks that sense DNA
damage and coordinate DNA replication, cell cycle arrest, and DNA repair.

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 27
The above-mentioned Cdc25A degradation pathway also appears to induce the
transient intra-S-phase response. Here, IR-induced formation of DSBs triggers
degradation of Cdc25A, which in turn inhibits the S-phase promoting activity
of CDK2/cyclin E and induces the transient blockade of DNA replication,
which delays S-phase progression for several hours (227). As described above,
Cdc25A destruction involves the phosphorylation of Cdc25A on Ser-123 by
both CHK1 and CHK2 in response to IR, and on Ser-75 by CHK1 in response
to UV irradiation (99) . Supporting the involvement in the S-phase checkpoint
of ATM, its phosphorylation targets including CHK2, and the CHK2-regu-
lated Cdc25A-CDK2 cascade, is the fact that mutants of ATM, CDK2, or the
other proteins in the CHK2-regulated Cdc25A-CDK2 cascade fail to inhibit S-
phase progression when they are irradiated. Consequently, these cells undergo
radio-resistant DNA synthesis (RDS), which is a phenomenon of persistent
DNA synthesis after irradiation (127,199).
Another phosphorylation target of ATM, the master transducer of the S-
phase checkpoint, plays a key role in the intra-S-phase checkpoint, namely,
BRCA1 (breast cancer susceptibility gene 1). BRCA2 may also be an impor-
tant target of ATM (228,229). Mutations in the BRCA1andBRCA2tumor sup-
pressor genes are responsible for the great majority of familial breast and
ovarian cancers. These proteins form nuclear foci with Rad51 during S phase
and after DNA damage (230). BRCA1-andBRCA2-mutant cells exhibit de-
fects in the homologous repair of chromosomal DSBs. BRCA1orBRCA2defi-
ciency in mice results in early embryonic lethality, but conditional deletions
reveal that mice with BRCA1orBRCA2mutations suffer a wide range of carci-
nomas(231). Moreover, a mammary epithelium whose BRCA1orBRCA2gene
has been deleted is highly susceptible to mammary tumorigenesis (232) .
BRCA1 is omnipresent and plays broad roles in transcriptional regulation that
include both p53-dependent and -independent responses. It also has ubiquitin
ligase activity when dimerized to Bard1, and undergoes damage-associated
phosphorylation by multiple kinases that precedes repair-complex formation
(230). In contrast, BRCA2 has a more straightforward function{\}it is central
to homology-directed repair (HDR) because of its interaction with Rad51 and
its direct binding to single-stranded DNA (233).
Another important phosphorylation target of ATM that plays a role in the
intra-S-phase checkpoint is NBS1 (Nijmegen breakage syndrome gene 1) (234–
236). NBS 1 (Xrs2 in yeast) forms a multimeric complex with the MRE11/
RAD50 nuclease, MDC1 (mediator of DNA damage checkpoint protein 1),
and other unidentified proteins, and recruits them to the vicinity of DNA dam-
age sites by direct binding to the phosphorylated histone H2AX (237). ATM
phosphorylates NBS1 at Ser-343 in response to IR (238). Cells harboring a
point mutation of NBS1 at this phosphorylation site failed to engage in the S-

28 Nojima
phase checkpoint induced by IR (239). Moreover, in collaboration with the
BRCA1 C-terminus domain, the highly conserved NBS1 forkhead-associated
domain plays a crucial role in the recognition of damaged sites(240). After
recognizing the DNA damage, the NBS1 complex proceeds to rejoin the DSBs
predominantly by homologous recombination repair in vertebrates. This pro-
cess collaborates with the cell cycle checkpoints at S and G2 phase to facilitate
DNA repair.
Mutations in the MRE11-complex genes result in sensitivity to DNA dam-
age, genomic instability, telomere shortening, aberrant meiosis, and abnormal
checkpoint signaling in S phase. Blockade of NBS1-MRE11 function and the
CHK2-Cdc25A-CDK2 pathway entirely abolishes the inhibition of DNA syn-
thesis that is normally induced by IR. This results in the complete RDS that is
also seen when cells harbor a defective ATM gene (227). However, the phos-
phorylation of NBS1 and CHK2 by ATM seems to trigger two distinct branches
of the intra-S-phase checkpoint because CDK2-dependent loading of Cdc45
onto replication origins, a prerequisite for the recruitment of DNA polymerase,
is prevented in normal or NBS1/MRE11-defective cells when they are irradi-
ated but not in irradiated cells that harbor a defective ATM protein (227).
53BP1, which plays a partially redundant role in the phosphorylation of the
downstream checkpoint effector proteins BRCA1 and CHK2, is also a key
transducer of the intra-S-phase and G2-M checkpoint arrests that occur in re-
sponse to IR (241).
CHK1 may also be necessary for the intra-S-phase checkpoint when DNA
synthesis is inhibited by DNA damage (242). Supporting this is that chemical
or genetic ablation of human CHK1 triggers the accumulation of Cdc25A, pre-
vents the IR-induced degradation of Cdc25A, and causes RDS (87). Moreover,
the basal turnover of Cdc25A operating in unperturbed S phase requires CHK1-
dependent phosphorylation of its Ser-123, Ser-178, Ser-278, and Ser-292 resi-
dues(100). The ATR-CHK1 pathway may also play an important role in the
intra-S-phase checkpoint that is induced by replication-associated DSBs caused
by application of the topoisomerase I inhibitor topotecan (TPT) (243), although
it has no relationship with DNA-PK activity (244). However, in budding yeast,
the intra-S-phase checkpoint control is not activated by another topoisomerase
I inhibitor, camptothecin (CPT), and the CPT-hypersensitive mutant strain that
fails histone 2A (H2A) Ser-129 phosphorylation is an essential component for
the efficient repair of DSBs that do not induce the intra-S-phase checkpoint
(245). In Xenopusegg extracts, DNA lesions generated by exonuclease or
etoposide, a DNA topoisomerase II inhibitor, activate a DNA damage check-
point that blocks the initiation of DNA replication (246). TLK, a protein kinase
that is potentially involved in regulating chromatin assembly and that is phos-

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 29
phorylated by CHK1 on its Ser-695 residue, also appears to be involved in the
ATM/CHK1-dependent intra-S-phase checkpoint (172).
Besides its function with H2AX (a histone H2A variant), Mdc1 (mediator of
DNA damage checkpoint protein 1) controls damage-induced checkpoints by
promoting the recruitment of repair proteins to the sites of DNA breaks (247).
Cells that lack the MDC1gene are sensitive to IR because they fail to activate
the intra-S-phase and G2/M checkpoints properly, probably due to an inability
to regulate CHK1 properly. Thus, MDC1 facilitates the establishment of the
intra-S-phase cell cycle checkpoint (248). Notably, MDC1 is hyperphos-
phorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear
foci at sites of DNA damage, which appears to be crucial for the efficient acti-
vation of the intra-S-phase checkpoint (249).
The ATR/ATRIP complex requires the RFC (replication factor C) and
PCNA-like proteins to fully activate the replication-stress response because
RFC recognizes the primer-template junction and recruits PCNA onto DNA to
function as a sliding clamp that tethers DNA polymerases (4,250). In fission
yeast and humans, the PCNA-like complex (Rad1/Rad9/Hus1 or RAD1/RAD9/
HUS1) is recruited in a RAD17-dependent manner onto the chromatin after
damage(149,251). In budding yeast, the homologous PCNA-like complex
(Rad17/Mec3/Ddc1) is recruited to DSBs and the sites of DNA damage in a
Rad24-dependent manner (252,253). Thus, it is possible that the Rad17 com-
plex recognizes DNA damage and loads the PCNA-like complex onto DNA,
thereby responding to DNA damage independently of ATR/ATRIP (254).
As with fission yeast, RAD17 and HUS1 are required for the phosphoryla-
tion of CHK1 by ATR in mammals (254,255) . ATR also phosphorylates Rad17
at its Ser-635 and Ser-645 residues (256) . This phosphorylation is significantly
stimulated by the increased amounts of PCNA-like complexes that were re-
cruited onto the chromatin after damage. Unlike the hus1-null fission yeast
cells, which are defective for the G2/M DNA-damage checkpoint, mouse cells
that lack the mouse homolog of the fission yeast protein Hus1 enter mitosis
normally after DNA damage but display an S-phase checkpoint defect (257).
The mouse Hus1 protein also seems to play a role in the NBS1-independent
checkpoint-mediated inhibition of DNA synthesis that is generated by the
genotoxin benzo(a)pyrene dihydrodiol epoxide (BPDE), which causes bulky
DNA adducts. However, the hus1-null mouse cells displayed intact S-phase
checkpoint responses in response to IR-induced DSBs (257).
6. Defects in G1/S Checkpoint and Cancer
Defects in the genome maintenance mechanisms, including DNA repair and
cell cycle checkpoint pathways, are believed to enhance genetic instability and

30 Nojima
cause the accumulation of mutations and chromosomal aberrations that is a
hallmark of cancer cells (155). Most of the G1/S checkpoint transducers and
effectors are classified as either tumor suppressors or proto-oncogenes, and
their loss-of-function mutations or overexpression appear to play pivotal roles
in many types of human tumors. Mouse models that mimic the defects of these
genes display similar phenotypes to human patients, which suggests that these
checkpoint regulators are important in the surveillance of genomic destabiliza-
tion and the prevention of tumor development.
Mutations in the p53 gene are responsible for the large majority of sporadic
human cancers, and thus p53 is a key target for cancer therapy
(67,108,110,135). p53 gene mutations can also be inherited in a subset of fami-
lies with the Li-Fraumeni syndrome (LFS), which is characterized by a predis-
position to sarcomas, brain and breast tumors, and childhood adrenocortical
carcinoma(258). The inactivation of the INK4a/ARF(orCDKN2a) locus,
which engages the pRB and p53 tumor suppressor pathways through its capac-
ity to encode the two distinct gene products p16
INK4a
and p14
ARF
, is also a
common genetic event in the development of human melanoma (259). Human
cells harboring pRB and p53 mutations also cause telomere dysfunction that
results in the chromosomal end-end joining and fusion-bridge-breakage cycles
that trigger the aneuploidy observed in most cancer cells (67). Both p53- and
ARF-deficient mice spontaneously develop tumors and die of cancers early in
life, and the primary MEFs cultured from p53- and ARF-deficient mice do not
senesce in culture but instead yield immortal cell lines (67). Moreover, many
Burkitt lymphomas (BL) carry point mutations in the p53 tumor suppressor
gene, bear other defects in the p14
ARF
-MDM2-p53 pathway, or the p16
INK4a
gene is inactivated by promoter methylation or homozygous deletion (260).
Thus, disruption of both the pRB and p53 pathways is also critical for BL
development. Overexpression of cyclin E, which deregulates the G1/S check-
point and contributes to genomic instability, is also observed in several types
of human tumors, including carcinomas of the lung, breast, and head and neck
(21). Furthermore, overexpression of Cdc25A in a subset of breast cancers is
associated with poor patient survival, which suggests that both Cdc25A and its
downstream target CDK2 might represent suitable therapeutic targets in early-
stage breast cancer (261).
ATM is the gene responsible for the rare disorder A-T, which is a genomic
instability syndrome that causes cancer predisposition, radiation sensitivity,
neurodegeneration, and immunodeficiency. The cells of A-T patients show
markedly abnormal cell cycle checkpoint responses at G1, S, and G2
(127,199,262). Moreover, while LFS, the highly penetrant familial cancer phe-
notype, is usually associated with inherited mutations in the p53 gene, some
LFS families that do not have germline mutations of p53 have instead het-

G1 and S-Phase Checkpoints, Chromosome Instability, and Cancer 31
erozygous germline mutations in CHK2 (258,263). This suggests that human
CHK2 is a tumor suppressor gene whose mutation confers a predisposition to
sarcomas, breast cancers, and brain tumors. Supporting this is the fact that oc-
casional sporadic cancer-associated mutations have been detected in both the
CHK1 and CHK2 genes (263). In certain patients with an A-T-like disorder
(A-TLD), mutations in MRE11, but not in ATM, are found, and the clinical
presentations of these patients mutated in hMRE11genes are virtually identi-
cal to those seen in A-T patients (237,264).
Fanconi’s anemia (FA) is an autosomal recessive disease that is character-
ized by bone marrow failure, developmental anomalies, a high incidence of
myelodysplasia and acute nonlymphocytic leukemia, cellular hypersensitivity
to crosslinking agents, and a high risk of developing acute myeloid leukemia
and certain solid tumors (265,266). The six known FA gene products (FANCA,
FFANCC, FANCD2, FANCE, FANCF, and FANCG proteins) interact in a
common pathway, in which the mono-ubiquitination and nuclear foci forma-
tion of FANCD2 are essential. Mono-ubiquitinated FANCD2 colocalizes with
BRCA1 and hRad51 in S-phase-specific nuclear foci (265,267). ATM phos-
phorylates FANCD2 on its Ser-222 residue in response to IR, and this is re-
quired for the activation of an S-phase checkpoint. Thus, FANCD2 links the
FA and ATM damage-response pathways (268) . Consequently, the FA pro-
teins are involved in the cell cycle checkpoint and DNA-repair pathways, and
disruption of the FA genes results in chromosome instability, a common fea-
ture of many human cancers (232).
TheBRCA1 gene was cloned by positional cloning as one of the genes that
confers genetic predisposition to early-onset breast and ovarian cancer (230).
TheBRCA2tumor-suppressor gene was also identified by a similar approach
(230). Inherited mutations in BRCA1orBRCA2predispose people to develop
breast, ovarian, and other cancers (269).BRCA2has been identified as being
the seventh FA gene, and mutated BRCA2 protein fails to bind to Rad51 in
response to genotoxic stress, which prevents Rad51 from localizing to nuclear
damage foci (231,270). It has been suggested that thet FA proteins FANCA,
BRCA2, and FANCD2 act indirectly with the cellular defense machinery
against oxidative stress by linking it with the defense machinery against DNA
damage(271).
Nijmegen breakage syndrome (NBS) is a recessive genetic disorder that is
characterized by elevated sensitivity to IR that induces DSBs and a high fre-
quency of malignancies (240). Cells derived from NBS patients show chromo-
some fragility, IR sensitivity, and RDS (failure to suppress S-phase progression
in the presence of IR-induced DSBs) (239). These phenotypic features are remi-
niscent of those in the cells established from A-T patients, although the clinical
presentation of NBS differs considerably from that of A-T. RDS was first re-

32 Nojima
ported for cells derived from A-T patients and was later found in NBS, A-
TLD, and FA patients as well (266,269) . Moreover, cells derived from tumors
with mutated BRCA1(272)andCHK2(202)genes also undergo RDS when
they are irradiated. It has been proposed that in combination with defects in
other cell cycle checkpoints, RDS may contribute to the destabilization of the
genome, thereby predisposing individuals bearing these genetic aberrations to
cancer.
Patients with the rare genetic disease Bloom’s syndrome (BS) are predis-
posed to developing all the cancers that affect the general population. BS arises
through mutations in both alleles of the BLM (Bloom’s syndrome mutated)
gene, which encodes a 3'-5' DNA helicase, a member of the RecQ family. Cells
derived from BS patients exhibit marked genetic instability, and BLM protein
is known to contribute to the cellular response to IR by acting as a downstream
ATM kinase effector (273). Notably, BLM-deficient cells exhibit a normal p53
response to IR, as well as an intact G1/S cell cycle checkpoint, which indicates
that the ATM and p53 pathways are functional in BS cells (273). BLM-defi-
cient cells also exhibit an intact S-phase arrest, proper recovery from S-phase
arrest, and intact p53 and p21 responses after HU treatment. However, BLM-defi-
cient cells show a reduction in the number of replicative cells and a partial escape
from the G2/M cell cycle checkpoint, and have an altered p21 response (274).
Many tumors display numerical and structural centrosome aberrations. Re-
cent evidence shows that the centrosome plays an active role not only in the
regulation of microtubule nucleation activity, but also in the coordination of
centrosome duplication with cell cycle progression, in the stress response, and
in cell cycle checkpoint control (275). The single centrosome in G1 phase is
duplicated during S phase. The two centrosomes then set up the poles of the
mitotic spindle, and each incipient daughter cell receives one centrosome (276).
Note that centrosome aberrations can give rise to chromosomal instability, and
cells that lack a functional p53 pathway are proposed to acquire multiple cen-
trosomes through the failure of a G1-phase checkpoint (277). p53 controls cen-
trosome duplication by either direct physical binding to the centrosomes or by
enhancing p21
WAF1
expression, which regulates the timely activation of CDK2/
cyclin E and ensures the coordinated initiation of centrosome and DNA dupli-
cation(277). Thus, loss or mutational inactivation of p53 leads to abnormal
amplification of centrosomes due to the deregulation of the centrosome dupli-
cation cycle, which increases the frequency of mitotic defects and unbalanced
chromosome transmission to daughter cells.
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Title: A Brief Bible History: A Survey of the Old and New
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*** START OF THE PROJECT GUTENBERG EBOOK A BRIEF BIBLE
HISTORY: A SURVEY OF THE OLD AND NEW TESTAMENTS ***

A BRIEF
BIBLE HISTORY
A SURVEY OF THE OLD AND NEW TESTAMENTS
JAMES OSCAR BOYD, Ph.D., D.D.
AND
JOHN GRESHAM MACHEN, D.D.
PHILADELPHIA
THE WESTMINSTER PRESS
1922
Coéyright , 1922, by
The Truëteeë of the Preëbyterian Board of
Publication and Sabbath School Work
Printed in the United States of America

Contents
Section I
THE DEVELOPMENT OF THE CHURCH IN OLD TESTAMENT TIMES
Leëëon Page
I.Before Abraham 7
II.The Patriarchs 10
III.Egyptian Bondage and Deliverance 13
IV.Moses as Leader and Lawgiver 16
V.The Conquest and Settlement of Canaan19
VI.The Period of the Judges 22
VII.Samuel and Saul: Prophecy and Monarchy25
VIII.David and Solomon: Psalms and Wisdom28
IX.The Kingdom of Israel 31
X.The Kingdom of Judah, to Hezekiah 34
XI.Judah, from Hezekiah to the Exile 37
XII.The Exile and the Restoration 40
XIII.The Jewish State Under Persia 43
XIV.Israel's Religious Life 46
XV."The Coming One" 49
Section II
THE LIFE OF CHRIST AND THE DEVELOPMENT OF THE CHURCH IN
NEW TESTAMENT TIMES
I.The Preparation 55
II.The Coming of the Lord 58

III.The Baptism 61
IV.The Early Judean Ministry 64
V.The Beginning of the Galilæan Ministry 67
VI.The Period of Popularity 70
VII.The Turning Point 73
VIII.Jesus as Messiah 76
IX.The Prediction of the Cross 79
X.The Last Journeys 83
XI.Teaching in the Temple 86
XII.The Crucifixion 89
XIII.The Resurrection 93
XIV.The Beginnings of the Christian Church 96
XV.The First Persecution 99
XVI.The Conversion of Paul 102
XVII.The Gospel Given to the Gentiles 105
XVIII.The First Missionary Journey and the Apostolic Council109
XIX.The Second Missionary Journey 112
XX.
The Third Missionary Journey. The Epistle to the
Galatians
115
XXI.
The Third Missionary Journey. The Epistles to the
Corinthians and to the Romans
118
XXII.The First Imprisonment of Paul 122
XXIII.The Close of the Apostolic Age 125

Introduction
This book surveys the history of God's redeeming grace. It reviews
Old Testament history, disclosing the stream of God's redeeming
purposes flowing down through the older times. It reviews New
Testament history, disclosing the broadening and deepening of that
purpose for us men and for mankind in our Lord and Saviour Jesus
Christ and his Church.
The chapters included in this book appear also as a part of Teaching
the Teacher, a First Book in Teacher Training, and are issued in this
form to supply the demand for a brief Bible history, for popular
reading.
Harold McA. Robinëon .

SECTION I
The Development of the Church in Old Testament Times
By James Oscar Boyd, Ph.D., D.D.

LESSON I
Before Abraham
Genesis, Chapters 1 to 11
That part of the globe which comes within the view of the Old
Testament is mostly the region, about fifteen hundred miles square,
lying in the southwestern part of Asia, the southeastern part of
Europe, and the northeastern part of Africa. This is where the three
continents of the Eastern Hemisphere come together. Roughly
speaking it includes Asia Minor, Mesopotamia, Syria, Palestine,
Arabia, and Egypt, with a fringe of other lands and islands stretching
beyond them.
The heart of all this territory is that little strip of land, lying between
the desert on the east and the Mediterranean Sea on the west,
known as Syria and Palestine. It is some four hundred miles in
length and varies from fifty to one hundred miles in width. It has
been well called "the bridge of the world," for like a bridge it joins
the largest continent, Asia, to the next largest, Africa. And as
Palestine binds the lands together, so the famous Suez Canal at its
southern end now binds the seas together. To-day, therefore, as in
all the past, this spot is the crossroads of the nations.
Palestine has long been called the "Holy Land," because it is the
scene of most of the Bible story. Yet it would be a mistake to
suppose that that Bible story is limited to Palestine. The book of
Genesis does not introduce the reader to Canaan (as it calls
Palestine) until he has reached its twelfth chapter. There is a sense
in which the history of God's people begins with Abraham, and it
was Abraham who went at God's bidding into the land of Canaan.
The story of Abraham will be taken up in the second lesson; but the

Bible puts before the life of Abraham all the familiar story that lies in
the first eleven chapters of Genesis and that forms the background
for the figures of Abraham and his descendants.
The location of this background is the basin of the Tigris and
Euphrates Rivers. These two streams are mentioned in Gen. 2:14
(the Tigris under the form "Hiddekel") as the third and fourth
"heads" of the "river that went out of Eden to water the garden" in
which our first parents dwelt. The region is at the southern end of
what is now called Mesopotamia. At the northern end of this river
basin towers the superb mountain known as Mount Ararat. But the
"mountains of Ararat," mentioned in Gen. 8:4 as the place where
Noah's ark rested when the waters of the Flood had subsided, are no
particular peak, but are the highlands of Kurdistan, which in ancient
times were called Urartu (Ararat). Between Kurdistan on the north
and the Persian Gulf on the south, the highlands of Persia on the
east and the great Syrian Desert on the west, occurred the earliest
drama of human history.
That drama was a tragedy. It became a tragedy because of man's
sin. The wonderful poem of creation in Gen., ch. 1, has for the
refrain of its six stanzas, "God saw that it was good." Best of all was
man, the last and highest of God's works—man, made in "his own
image," after his likeness. On the sixth "day," when God made man,
God said of his work, "Behold, it was very good." More than that:
through the kindness of God man is put in a "garden," and is
ordered to "dress it and to keep it." Ch. 2:15. Adam sees his
superiority to the rest of the animal kingdom, over which he is given
"dominion." He is thus prepared to appreciate the woman as a
helpmeet for him, so that the unit of society may ever mean for him
one man and one woman with their children. Adam is also warned
against sin as having disobedience for its root and death as its
result.
All this prepares us to understand the temptation, the miserable fall
of the woman and the man, their terror, shame, and punishment.
Ch. 3. And we are not surprised to see the unfolding of sin in the life

of their descendants, beginning with Cain's murder of Abel, and
growing until God sweeps all away in a universal deluge. Chs. 4, 6.
God's tender love for his foolish, rebellious creatures "will not let
them go." At the gates of the garden from which their sin has
forever banished them, God already declares his purpose to "bruise"
the head of that serpent, Rom. 16:20, who had brought "sin into the
world and death by sin," Gen. 3:15. Through the "seed of the
woman"—a "Son of man" of some future day—sinful man can
escape the death he has brought upon himself. And from Seth, the
child "appointed instead of" murdered Abel, a line of men descends,
who believe this promise of God. Ch. 5. In Enoch we find them
"walking with God," v. 24, in a fellowship that seemed lost when
paradise was lost. In Lamech we find them hoping with each new
generation that God's curse will be at length removed. V. 29. And in
Noah we find them obedient to a positive command of God, ch.
6:22, as Adam had been disobedient.
In the Flood, Noah and his family of eight were the only persons to
survive. When they had come from the ark after the Flood, God gave
them the promise that he would not again wipe out "all flesh." Ch.
9:11. But after it appeared that God's judgments had not made
them fear him, God was just as angry with Noah's descendants as
he had been with the men before the Flood. Pride led them to build
a tower to be a rallying point for their worship of self. But God
showed them that men cannot long work together with a sinful
purpose as their common object; he broke up their unity in sin by
confusing their speech, ch. 11, and scattering them over the earth,
ch. 10. This second disappointment found its brighter side in the line
of men descended from Noah through Shem, ch. 11:10, who also
cherished God's promises. And the last stroke of the writer's pen in
these earliest chapters of the Bible introduces the reader to the
family of Terah in that line of Shem, and thus prepares the way for a
closer acquaintance with Terah's son, Abraham, "the friend of God."
QUESTIONS ON LESSON I
1. About how large is the world of the Old Testament, and where does it lie?

2. What special importance has Palestine because of its position?
3. How much of the story in Genesis is told before we are carried to
Palestine?
4. Locate on a map the scene of those earliest events in human history.
5. Show how the first two chapters of Genesis prepare for the tragedy of sin
and death that follows.
6. How does the brighter side of hope and faith appear from Adam to Noah?
7. What effect did the Flood have on men's sin and their faith in God?
8. Trace the descent of the man God chose to become "the father of the
faithful."

LESSON II
The Patriarchs
Genesis, Chapters 12 to 50
God's purpose to save and bless all mankind was to be carried out in
a wonderful way. He selected and "called" one man to become the
head and ancestor of a single nation. And in this man and the nation
descended from him, God purposed to bless the whole world.
Abraham was that man, and Israel was that nation. God made
known his purpose in what the Bible calls the Promise, Gal. 3:17, the
Blessing, v. 14, or the Covenant, v. 17. Its terms are given many
times over in the book of Genesis, but the essence of it lies already
in the first word of God to Abraham, Gen. 12:3, "In thee shall all the
families of the earth be blessed."
To believe this promise was a work of faith. It was against all
appearances and all probability. Yet this was just where the religious
value of that promise lay for Abraham and for his children after him
—in faith. They had to believe something on the basis solely of their
confidence in the One who had promised it. Or rather, they had to
believe in that Person, the personal Jehovah, their God. They must
absolutely trust him. To do so, they must "know him." And that they
might know him, he must reveal himself to them. That is why we
read all through Genesis of God's "appearing" or "speaking" to this
or the other patriarch. However he accomplished it, God was always
trying thus to make them better acquainted with himself; for such
knowledge was to be the basis of their faith. Upon faith in him
depended their faith in his word, and upon faith in his word
depended their power to keep alive in the world that true religion

which was destined for all men and which we to-day share.
Abraham's God is our God.
Not Abraham's great wealth in servants, Gen. 14:14, and in flocks
and herds, ch. 13:2, 6, but the promise of God to bless, constituted
the true "birthright" in Abraham's family. Ishmael, the child of doubt,
missed it; and Isaac, the child of faith, obtained it. Gal. 4:23. Esau
"despised" it, because he was "a profane [irreligious] person," Heb.
12:16, and Jacob schemed to obtain it by purchase, Gen. 25:31, and
by fraud, ch. 27:19. Jacob bequeathed it to his sons, ch. 49, and
Moses delivered it in memorable poetic form to the nation to retain
and rehearse forever. Deut., ch. 32.
When Abraham, the son of Terah, entered Canaan with Sarah his
wife and Lot his nephew and their great company of servants and
followers, he was obeying the command of his God. He no sooner
enters it than God gives him a promise that binds up this land with
him and his descendants. Gen. 13:14-17. Yet we must not suppose
that Abraham settled down in this Promised Land in the way that the
Pilgrim Fathers settled in the Old Colony. Although Canaan is
promised to the "seed" of Abraham, Isaac, and Jacob as a
possession, they did not themselves obtain a foothold in it. Apart
from the field of the cave Machpelah, at Hebron in the south, Gen.,
ch. 23, and a "shoulder" (shechem) or fragment of land near
Shechem ("Jacob's Well"), in the center of Canaan, the patriarchs
did not acquire a foot of the soil of what was to become "the Holy
Land." Abraham wandered about, even going down to Egypt and
back. Isaac was sometimes at Hebron and sometimes at Beer-sheba
on the extreme southern verge of the land. Jacob spent much of his
manhood in Mesopotamia, and of his old age in Egypt. For after
divine Providence in a remarkable manner had transplanted one of
Jacob's sons, Joseph, into new soil, Gen., ch. 37, his father and his
brothers were drawn after him, with the way for their long Egyptian
residence providentially prepared for them, Gen. 50:20.
Side by side with the growth of a nation out of an individual we find
God's choice of the direction which that growth should take. Not all,

even of Abraham's family, were to become part of the future people
of God. So Lot, Abraham's nephew, separates from him, and
thereafter he and his descendants, the Ammonites and the
Moabites, go their own way. As between Abraham's sons, Ishmael is
cast out, and Isaac, Sarah's son, is selected. And between Isaac's
two sons, Esau and Jacob, the choice falls on Jacob. All twelve of
Jacob's sons are included in the purpose of God, and for this reason
the nation is called after Jacob, though usually under his name
"Israel," which God gave him after his experience of wrestling with
"the angel of the Lord" at the river Jabbok. Gen. 32:22. Those sons
of his are to become the heads of the future nation of the "twelve
tribes", Acts 26:7.
Even while Lot, Ishmael, and Esau are thus being cut off, the
greatest care is taken to keep the descent of the future nation pure
to the blood of Terah's house. Those three men all married alien
wives: Lot probably a woman of Sodom, Ishmael an Egyptian, and
Esau two Hittite women. The mother of Isaac was Sarah, the mother
of Jacob was Rebekah, and the mothers of eight of the twelve sons
of Jacob were Leah and Rachel; and all these women belonged to
that same house of Terah to which their husbands belonged. Indeed,
much of Genesis is taken up with the explanation of how Isaac and
Jacob were kept from intermarrying with the peoples among whom
they lived.
The last quarter of the book, which is occupied with the story of
Joseph and his brethren, is designed to link these "fathers" and their
God with the God and people of Moses. The same Jehovah who had
once shown his power over Pharaoh for the protection of Abraham
and Sarah, and who was later to show his power over another
Pharaoh "who knew not Joseph," showed his power also over the
Pharaoh of Joseph's day, in exalting Joseph from the dungeon to the
post of highest honor and authority in Egypt, and in delivering Jacob
and his whole family from death through Joseph's interposition.
What their long residence in Egypt meant for God's people will be
seen in another lesson.

QUESTIONS ON LESSON II
1. In what promise does God reveal to Abraham his plan to bless the world?
2. How was Abraham brought to believe in God's promise? What difference
did it make whether he and his descendants believed it or not?
3. Did the patriarchs see that part of the promise fulfilled which gave them
possession of "the Holy Land"? Read carefully Gen. 15:13-16 and Heb.
11:9, 10, 14-16.
4. Make a "family tree" in the usual way, showing those descendants of Terah
who play any large part in the book of Genesis. Underscore in it the
names of those men who were in the direct line of "the Promise."
5. How were Isaac and Jacob kept from marrying outside their own family?
6. Explain Joseph's words, "Ye meant evil against me; but God meant it for
good, to bring to pass, as it is this day, to save much people alive." Gen.
50:20.

LESSON III
Egyptian Bondage and Deliverance
Exodus, Chapter 1
God says through his prophet Hosea, Hos. 11:1, "When Israel was a
child, then I loved him, and called my son out of Egypt." See also
Matt. 2:15. There was a loving, divine purpose in the Egyptian
residence of God's people. What was it? What did this period mean
in the career of Israel?
Most obviously, it meant growth. From the "seventy souls," Ex. 1:5,
that went down into Egypt with Jacob, there sprang up there a
populous folk, large enough to take its place alongside the other
nations of the world of that day. Observe the nature of the land
where this growth took place. Egypt was a settled country, where
the twelve developing tribes could be united geographically and
socially in a way impossible in a country like Palestine. However
oppressed they were, they nevertheless were secluded from the
dangers of raids from without and of civil strife within—just such
dangers as later almost wrecked the substantial edifice slowly
erected by this period of growth in Egypt.
Egypt meant also for Israel a time of waiting. All this growth was not
accomplished in a short time. It lasted four hundred and thirty years.
Ex. 12:40, 41. Through this long period, which seems like a dark
tunnel between the brightness of the patriarchs' times and that of
Moses' day, there was nothing for God's people to do but to wait.
They were the heirs of God's promise, but they must wait for the
fulfillment of that promise in God's own time, wait for a leader raised
up by God, wait for the hour of national destiny to strike. As Hosea,
ch. 11:1 expresses it, this "child" must wait for his Father's "call."

The Egyptian period left an indelible impression on the mind of
Israel. It formed the gray background on which God could lay the
colors of his great deliverance. It is because God knew and planned
this that he so often introduces himself to his people, when he
speaks to them, as "Jehovah thy God, who brought thee out of the
land of Egypt, out of the house of bondage."
In the third place, this Egyptian period meant for Israel a time of
chastisement. The oppression to which the descendants of Jacob
were exposed, when "there arose a new king over Egypt, who knew
not Joseph," Ex. 1:8, was so severe, prolonged, and hopeless, v. 14,
that it has become proverbial and typical. Since every male child was
to be put to death, v. 22, it is clear that the purpose of the Egyptians
was nothing less than complete extermination. "It is good for a man
that he bear the yoke in his youth": if that be true, then the children
of Israel derived good from the school of discipline in which they
grew up. True, as we read their later story, we feel that no people
could be more fickle. Yet there is no other nation with which to
compare Israel. And it is very probable that no other nation would
have been serious-minded enough even to receive and grasp the
divine revelation and leading of Moses' and Joshua's time. God, who
had "seen the affliction of his people," who had "heard their cry" and
sent Moses to them to organize their deliverance, wrote forever on
this nation's soul the message of salvation in a historical record. At
the start of their national life there stood the story, which they could
never deny or forget, and which told them of God's power and
grace.
Exodus, Chapters 5 to 15
All this lay in Israel's experience in Egypt. The next lesson will tell of
the character and work of the man whom God chose to be leader.
The means by which Moses succeeded in the seemingly impossible
task of marching a great horde of slaves out from their masters'
country, was the impression of God's power on the minds of Pharaoh
and his people. It was a continued, combined, and cumulative
impression. Of course it could not be made without the use of

supernatural means. We must not, therefore, be surprised to find
the story in Exodus bristling with miracles. To be sure, the "plagues"
can be shown to be largely natural to that land where they occurred.
And the supreme event of the deliverance, the passage of Israel
through the Red Sea on dry ground, was due, according to the
narrative itself, to a persistent, wind, Ex. 14:21, such as often lays
bare the shallows of a bay, only to release the waters again when its
force is spent.
Nevertheless, it is not possible to remove the "hand of God" from
the account by thus pointing out some of the means God used to
accomplish his special purposes. It was at the time, in the way, and
in the order, in which Moses announced to Pharaoh the arrival of the
plagues, that they actually appeared. This was what had its ultimate
effect on the king's stubborn will. And when Israel was told to "go
forward," with the waters right before them, and when the Egyptians
were saying, "They are entangled in the land, the wilderness hath
shut them in," Ex. 14:3—it was just at that juncture that the east
wind did its work at God's command; when Israel was over safely, it
went down. Such things do not "happen." It made a profound
impression on Israel, on Egypt, and on all the nations of that day; all
united in accepting it as the work of Israel's God. Ex. 15:11, 14-16;
Josh. 2:10.
The important point for the nation was to know, when Moses and
Aaron came to them in the name of God, that it was their fathers'
God who had sent them. On account of this need, which both the
people and their leaders felt, God proclaimed his divine name,
Jehovah (more precisely, Yahweh, probably meaning "He is," Ex.
3:14, 15), to Moses, and bade him pronounce the same to Israel, to
assure them that he was "the God of Abraham, of Isaac, and of
Jacob," and thus what Moses came now to do for them was just
what had been promised to those fathers long before. The passover
night was the fulfillment of God's good word to Abraham. Ex. 13:10,
11. How that word went on and on toward more and more complete
fulfillment will be the subject of the succeeding lessons.

QUESTIONS ON LESSON III
1. What advantages had Egypt over Palestine as the place for Israel to grow
from a family into a nation?
2. What value was there for Israel in a negative time of waiting at the
beginning of its history?
3. Compare the effect on Israel with the effect on a man, of passing through
a time of difficulty while developing.
4. Name the ten "plagues of Egypt" in their order. How far can they be called
"natural"?
5. If the east wind drove back the Red Sea, what did God have to do with
Israel's escape from the Egyptian army?
6. Why should we not be surprised to find many miracles grouped at this
stage of Bible history?
7. How did God identify himself in the minds of the people with the God of
their fathers? What was his personal name?

LESSON IV
Moses as Leader and Lawgiver
Exodus, Chapters 2 to 4
One of the things Israel had to wait for through those centuries in
Egypt was a leader. When the time came God raised up such a
leader for his people in Moses.
The story of how Moses' life was preserved in infancy, and of how he
came to be brought up at the court of Pharaoh with all its
advantages for culture, is one of the most fascinating tales of
childhood. Ex. 2:1-10. But not many who know this familiar tale
could go on with the biography of the man of forty who fled from
Pharaoh's vengeance. Moses found by personal contact with his
"brethren," the children of Israel, that they were not yet ready for
common action, and would not easily acknowledge his right to lead
them. After killing an Egyptian slave driver there was nothing for
Moses to do but to flee. Vs. 11-15.
He spent the second forty years of his life, Acts 7:23, 30; Ex. 7:7, in
the deserts about the eastern arm of the Red Sea—the region
known to the Hebrews as Midian. There he married the daughter of
the Midianite priest Reuel. (Jethro was probably Reuel's title,
meaning "his excellency.") While herding his sheep in the mountains
called Horeb (Sinai), Moses received at the burning bush that
personal revelation of the God of his fathers, which lay at the base
of all his future labors for God and his people. Ex. 3:1 to 4:17. It was
a commission to lead Israel out of their bondage in Egypt into the
land promised to their fathers.

Though very humble as to his fitness for such leadership, Moses was
assured of Jehovah's presence and help. He was equipped with
extraordinary powers for convincing the proud Pharaoh that his
demands were God's demands; and he was given the aid of his
brother Aaron, who had a readiness of speech which Moses at this
time seems to have lacked.
Exodus, Chapters 16 to 24
How the two brothers achieved the seemingly impossible task of
winning out of Egypt, and of uniting a spiritless and unorganized
mass of slaves upon a desperate enterprise, is the narrative that fills
the early chapters of Exodus. But with Israel safe across the Red
Sea, Moses' leadership had only begun. He instituted an organization
of the people for relieving himself of his heavy duties as judge. He
determined the line of march, and sustained the spirits of the
fighting men in their struggle against the tribes of the desert who
challenged Israel's passage.
But, above all, Moses became the "mediator" of the "covenant,"
Heb. 9:19-21, between the Hebrews and Jehovah their God at
Mount Sinai. On the basis of the Ten Commandments, Ex. 20:2-17;
Deut. 5:6-21, that guide to God's nature and will which formed the
Hebrew constitution, the people agreed to worship and obey
Jehovah alone, and Jehovah promised to be their God, fulfilling to
them his promises made to their fathers. By solemn sacrifices,
according to the custom of the time, when the symbolism of altar
and priesthood was well understood, this covenant was sealed.
Exodus, Chapter 25 to Numbers, Chapter 36
After long seclusion on the mount alone with God, Moses ordered
the erection of a house of worship. It had to be portable, so as to
accompany them in their wanderings and express visibly, wherever
set up, the religious unity of the twelve tribes. Aaron and his sons
were consecrated to be the official priesthood of this new shrine and

were clothed and instructed accordingly. Minute details regulated all
sacrifices, and similar minute instructions enabled the priests to
decide questions of ceremonial cleanness and uncleanness in
matters of food and health.
All these laws and regulations, mainly recorded in Leviticus, were
given through Moses, either alone or in association with his brother.
It is not surprising to learn that there were those who challenged
this exclusive leadership in every department of the national life. We
read of a willful disregard of divine orders even in the family of
Aaron, with immediate fatal results. Lev. 10:1-7. Like punishment
overtook those members of the tribe of Levi who showed jealousy of
the house of Aaron, and those elements in other tribes that claimed
rights equal or superior to those of Moses. Num., chs. 16, 17. It
would be strange, indeed, if God, who had vindicated his servant
Moses against Pharaoh, should let his own authority as represented
by Moses be challenged within the camp of Israel. He punished to
save.
Just as God took up the Sabbath and circumcision, old customs of
the preceding era, into the law of Israel, so also he spoke to this
people through an elaborate system of feasts and pilgrimages, which
bound up their whole year with the worship of God. Indeed, the
principle of the seventh part of time as sacred was extended to the
seventh year, and even to the fiftieth year (the year following the
seventh seven), for beneficent social and economic uses. Lev., ch.
25.
When at length the nation, thus organized and equipped, set forth
from Sinai, Num. 10:11, they required a leadership of a different
kind—military leadership and practical statesmanship. They found
both in Moses. He it was who led them through all the long
wanderings in the peninsula of Sinai, bearing their murmurings and
meeting their recurrent difficulties with a patience that seems almost
divine, save for that one lapse which was to cost him and Aaron
their entrance into the Promised Land. Num. 20:10-12.

At the border of the land, from the top of Pisgah in the long
mountain wall of Moab, Moses at last looked down into that deep
gorge of the Jordan Valley at his feet, which separated him from the
hills of Canaan. Beyond this river and the Dead Sea, into which it
empties, lay the land long ago promised to the seed of Abraham.
Moses had been permitted to lead the people to its very gateway;
but it remained for another, his younger helper, Joshua, to lead them
through the gate into the house of rest.
The Book of Deuteronomy
But before he surrendered his power to another and his life to his
Maker, the aged Moses rehearsed in the ears of Israel the great
principles of God's law. He pleaded earnestly with them to accept it
from the heart, to adapt it to the changed conditions of their new
settled life with its new temptations, and to hand it down as their
most precious heritage to their children after them. This is the
purpose and substance of the book of Deuteronomy, which gets its
name from the fact that it is a "second lawgiving." It is the Law of
Sinai repeated, but in oratorical form, charged with the feeling and
spirit of that "man of God," whose name is forever linked with the
Law and with the God who gave it to mankind.
QUESTIONS ON LESSON IV
1. How did Moses' forty years in Egypt and his forty years in Midian help to
prepare him for leadership?
2. What was the constitution of the new Hebrew State established at Sinai?
How was it ratified?
3. How was the tabernacle suited to the religious needs of Israel during
Moses' lifetime?
4. Show how the Law of Moses takes up the old principle of the Sabbath and
applies it to the life of Israel.
5. Where did Moses' leadership end, and what was his last service to the
nation?

LESSON V
The Conquest and Settlement of Canaan
The Book of Joshua
On the death of Aaron his son, Eleazar, succeeded him as high
priest. But when Moses died, it was not a son who succeeded him in
the political and moral leadership of Israel, for that position was not
hereditary. Joshua, a man of Ephraim, was divinely designated for
this work. He was fitted for the difficult undertaking by military
experience, Ex. 17:9-14, by personal acquaintance with Canaan,
Num. 13:8, 16; 14:6, 30, 38, and by long and intimate association
with Moses, Ex. 33:11; Num. 11:28; Deut. 34:9; Josh. 1:1. The book
of Joshua, which records his career, divides naturally into two parts,
first, the conquest, chs. 1 to 12, and second, the settlement, chs. 13
to 22. Two further chapters, chs. 23, 24, contain Joshua's valedictory
address.
Before Moses' death two and a half tribes had already received their
assignment of territory on the east of the Jordan, out of lands
conquered from the Amorite kings, Sihon and Og. But the fighting
men of these tribes agreed to accompany the other tribes and share
their struggle till all had obtained an inheritance. So when the great
host passed over the Jordan, not far from where it empties into the
Dead Sea, the men of Reuben, Gad, and Manasseh crossed with the
rest. Jehovah, who at the Red Sea a generation earlier had struck
terror into the hearts of all nations by his wonderful interposition to
save Israel and destroy its enemies, repeated here his saving help,
by stemming the swift current of the Jordan River, till all had passed
over dry shod to the western side.

Once over, they found themselves face to face with Jericho, a city
which commanded the passes into the mountain country beyond.
Spies previously despatched to learn the weakness of Jericho had
reported the panic of its inhabitants and so prepared the Hebrews to
believe God's word, when through Joshua he announced a bloodless
victory here at the beginning of their conquest. Without a blow
struck Jericho fell, and all its inhabitants were "devoted," at
Jehovah's strict command. Even their wealth was to be "devoted,"
that is, the cattle slain and the goods added to the treasury of the
sanctuary. Only Rahab, who had saved the spies, and her family
were excepted. One man, Achan, disobeyed the ban on private
spoils. His covetousness and deception, revealed by Israel's defeat in
the expedition against Ai which followed the fall of Jericho, and
detected by the use of the sacred lot, was punished by the execution
of all who were privy to the crime.
Better success attended the second attempt to take Ai. With these
two cities reduced, Jericho at the bottom and Ai at the top of the
valley leading up from the Jordan floor to the central highland,
Joshua was in a position to attack anywhere without fear of being
outflanked. Middle, south, and north was the order commended by
military considerations. Accordingly those cities which, because in
the middle of the land, felt themselves the most immediately
threatened, took the first steps to avert the menace. A group of five
towns lying just north of Jerusalem, with Gibeon at their head,
succeeded by a ruse in getting a treaty of peace from Joshua. The
Gibeonites deceived Joshua by representing themselves as having
come from a great distance to seek an alliance. Joshua's pride was
flattered and he fell a victim to the trick. The consequences were
serious, for these Canaanites, though reduced to vassalage,
remained as aliens in the heart of the land, and cut off the southern
from the northern tribes of Israel.
A confederacy of the chief cities in the region south of Gibeon,
headed by the king of Jerusalem, determined to strike the first blow.
But their campaign against the Gibeonites, now the allies of Israel,
ended in a quick advance by Joshua and his complete subjugation of

all these cities, the humiliation and death of their kings, and the
"devotion" of the inhabitants who fell into his hands.
A similar campaign followed in the north, with the city of Hazor at
the head of the Canaanite forces. At the "waters of Merom," a small
lake a few miles north of the Sea of Galilee, a surprise attack by
Joshua deprived his enemies of their advantage in horsemen and
chariots on the level ground they had selected for battle, and
resulted in the utter rout of the Canaanites and the general
slaughter of every soul that did not escape by flight from the
"devoted" towns.
Thus from Mount Hermon on the north to the wilderness of the
wandering on the south, the whole land had been swept over and
reduced to impotence by the Hebrew invader. It was time to
apportion it now to the several tribes. This was accomplished under
the direction of Joshua and Eleazar. Judah and Joseph, the two
strongest tribes, were assigned, the one to the south and the other
to the north of the main mountain mass. Levi's inheritance was to be
"the Lord," that is, the religious tithes, and his dwelling was to be
"among his brethren," that is, in designated towns throughout all the
land. A commission of three representatives from each of the seven
other western tribes divided the rest of the conquered territory into
seven fairly equal parts. These then were assigned to the seven
tribes by lot at the tabernacle at Shiloh. As for the eastern tribes,
when they returned to their homes across the Jordan, they built an
altar at the ford, as a permanent "witness" to the unity of all the
sons of Jacob, however the deep gorge of the Jordan might cut
them off from one another.
At Shechem, where Abraham built his first altar in Canaan, Joshua
had renewed the covenant between the people and their God as
soon as he had secured control of Mount Ephraim, the middle
highlands. He had not only read the Law of Moses to all the people
here, but also inscribed it on stones for the sake of permanence and
publicity. And now, when the conquest was complete and Joshua
was nearing his end, he reassembled the people at the same spot,

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Checkpoint Controls And Cancer Volume 1 Reviews And Model Systems 1st Edition Hiroshi Nojima Auth

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