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About This Presentation
Biochem
Slide Content
© 2018 Cengage Learning. All Rights Reserved.
Chapter 10
Biosynthesis of Nucleic
Acids: Replication
Chapter 10
Biosynthesis of Nucleic
Acids: Replication
© 2018 Cengage Learning. All Rights Reserved.
Chapter Outline
(10-1) The flow of genetic information in the cell
(10-2) Replication of DNA
(10-3) DNA polymerase
(10-4) Proteins required for DNA replication
(10-5) Proofreading and repair
(10-6) DNA recombination
(10-7) Eukaryotic DNA replication
Chapter Outline
(10-1) The flow of genetic information in the cell
(10-2) Replication of DNA
(10-3) DNA polymerase
(10-4) Proteins required for DNA replication
(10-5) Proofreading and repair
(10-6) DNA recombination
(10-7) Eukaryotic DNA replication
© 2018 Cengage Learning. All Rights Reserved.
Flow of Genetic Information in the Cell
•Sequence of bases in DNA encodes genetic
information
•Replication: Process of duplication of DNA
•Requires RNA
•Transcription: Process of formation of RNA on a
DNA template
•Base sequence of DNA is reflected in the base
sequence of RNA
•Translation: Process of protein synthesis
•Amino acid sequence of the protein reflects the
sequence of bases in the gene that codes for that
protein
Flow of Genetic Information in the Cell
•Sequence of bases in DNA encodes genetic
information
•Replication: Process of duplication of DNA
•Requires RNA
•Transcription: Process of formation of RNA on a
DNA template
•Base sequence of DNA is reflected in the base
sequence of RNA
•Translation: Process of protein synthesis
•Amino acid sequence of the protein reflects the
sequence of bases in the gene that codes for that
protein
© 2018 Cengage Learning. All Rights Reserved.
Flow of Genetic Information in the Cell
(continued)
•Retroviruses are those viruses in which RNA is the
genetic material rather than DNA
•Catalyzed by reverse transcriptase
•Reverse transcriptase: Enzyme that directs the
synthesis of DNA on an RNA template
•Central dogma of molecular biology
•Scheme used to describe the manner in which
information is transferred in a cell
Flow of Genetic Information in the Cell
(continued)
•Retroviruses are those viruses in which RNA is the
genetic material rather than DNA
•Catalyzed by reverse transcriptase
•Reverse transcriptase: Enzyme that directs the
synthesis of DNA on an RNA template
•Central dogma of molecular biology
•Scheme used to describe the manner in which
information is transferred in a cell
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.1 -Mechanisms for Transfer of
Information in the Cell
Figure 10.1 -Mechanisms for Transfer of
Information in the Cell
© 2018 Cengage Learning. All Rights Reserved.
Replication of DNA
•Naturally occurring DNA exists in single-stranded and
double-stranded forms, both of which can exist in
linear and circular forms
•Makes it difficult to generalize about all cases of DNA
replication
•Replication of circular double-stranded DNA is
discussed here
•Most of the details of the replication process were first
investigated in prokaryotes, particularly E. coli
Replication of DNA
•Naturally occurring DNA exists in single-stranded and
double-stranded forms, both of which can exist in
linear and circular forms
•Makes it difficult to generalize about all cases of DNA
replication
•Replication of circular double-stranded DNA is
discussed here
•Most of the details of the replication process were first
investigated in prokaryotes, particularly E. coli
© 2018 Cengage Learning. All Rights Reserved.
Challenges in DNA Replication
•Achievement of continuous unwinding and separation
of the two DNA strands
•Protection of unwound portions from the action of
nucleases that attack single-stranded DNA
•Synthesis of the DNA template from the 5′ to the 3′
end
•Template has one 5′ → 3′ and one 3′ → 5′ strand
•Guarding against errors in replication by ensuring
that the correct base is added to the growing
polynucleotide chain
Challenges in DNA Replication
•Achievement of continuous unwinding and separation
of the two DNA strands
•Protection of unwound portions from the action of
nucleases that attack single-stranded DNA
•Synthesis of the DNA template from the 5′ to the 3′
end
•Template has one 5′ → 3′ and one 3′ → 5′ strand
•Guarding against errors in replication by ensuring
that the correct base is added to the growing
polynucleotide chain
© 2018 Cengage Learning. All Rights Reserved.
Semiconservative Replication
•DNA replication involves
separation of the two
original strands and
production of two new
daughter strands using
the original strands as
templates
•Each daughter strand
contains one template
strand and one newly
synthesized strand
Semiconservative Replication
•DNA replication involves
separation of the two
original strands and
production of two new
daughter strands using
the original strands as
templates
•Each daughter strand
contains one template
strand and one newly
synthesized strand
© 2018 Cengage Learning. All Rights Reserved.
Semiconservative Replication (continued)
•Experiment
•E. coli bacteria were
grown with
15
NH
4Cl as
the sole nitrogen source
•All newly formed nitrogen
compounds became
labeled with
15
N
•Method used
•Density-gradient
centrifugation
Semiconservative Replication (continued)
•Experiment
•E. coli bacteria were
grown with
15
NH
4Cl as
the sole nitrogen source
•All newly formed nitrogen
compounds became
labeled with
15
N
•Method used
•Density-gradient
centrifugation
© 2018 Cengage Learning. All Rights Reserved.
Direction of Replication
•DNA double helix unwinds at a specific point called
an origin of replication
•Polynucleotide chains are synthesized in either both
or in one direction from the origin of replication
•DNA replication is bidirectional in most organisms
•At each origin of replication, there are two replication
forks
•Replication forks: Points at which new polynucleotide
chains are formed
Direction of Replication
•DNA double helix unwinds at a specific point called
an origin of replication
•Polynucleotide chains are synthesized in either both
or in one direction from the origin of replication
•DNA replication is bidirectional in most organisms
•At each origin of replication, there are two replication
forks
•Replication forks: Points at which new polynucleotide
chains are formed
© 2018 Cengage Learning. All Rights Reserved.
Direction of Replication (continued)
•θstructure
•Bubble, or eye, of newly synthesized DNA between
regions of the original DNA is a manifestation of the
advance of the two replication forks in opposite
directions
•Prokaryotes - One origin of replication and one bubble
•Eukaryotes - Several origins of replication and several
bubbles
•Bidirectional growth of polynucleotide chains
represents net chain growth
Direction of Replication (continued)
•θstructure
•Bubble, or eye, of newly synthesized DNA between
regions of the original DNA is a manifestation of the
advance of the two replication forks in opposite
directions
•Prokaryotes - One origin of replication and one bubble
•Eukaryotes - Several origins of replication and several
bubbles
•Bidirectional growth of polynucleotide chains
represents net chain growth
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.4 - Bidirectional Replication
Figure 10.4 - Bidirectional Replication
© 2018 Cengage Learning. All Rights Reserved.
DNA Polymerase
•Last nucleotide added to a
growing chain has a 3′-hydroxyl
group on the sugar
•Acts as a nucleophile by
attacking the phosphorus
adjacent to the sugar in the
incoming nucleotide, which has
a 5′-triphostpahte on its sugar
•Causes the elimination of
pyrophosphate and the
formation of a new
phosphodiester bond
DNA Polymerase
•Last nucleotide added to a
growing chain has a 3′-hydroxyl
group on the sugar
•Acts as a nucleophile by
attacking the phosphorus
adjacent to the sugar in the
incoming nucleotide, which has
a 5′-triphostpahte on its sugar
•Causes the elimination of
pyrophosphate and the
formation of a new
phosphodiester bond
© 2018 Cengage Learning. All Rights Reserved.
Modes of Polymerization
•Leading strand
•Synthesized continuously from its 5′ end to its 3′ end at
the replication fork on the exposed 3′ to 5′ template
strand
•Lagging strand
•Synthesized semidiscontinuously in small fragments,
or Okazaki fragments
•5′ end of each fragment is closer to the replication fork
than the 3′ end
•Fragments are linked together by the enzyme DNA
ligase
Modes of Polymerization
•Leading strand
•Synthesized continuously from its 5′ end to its 3′ end at
the replication fork on the exposed 3′ to 5′ template
strand
•Lagging strand
•Synthesized semidiscontinuously in small fragments,
or Okazaki fragments
•5′ end of each fragment is closer to the replication fork
than the 3′ end
•Fragments are linked together by the enzyme DNA
ligase
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.6 - Semidiscontinuous Model for DNA
Replication
Figure 10.6 - Semidiscontinuous Model for DNA
Replication
© 2018 Cengage Learning. All Rights Reserved.
Example 10.1 -DNA Structure
•A nucleoside derivative that has been very much in
the news is 3′-azido-3′-deoxythymidine (AZT), as
shown in Figure 10.7
•This compound has been widely used in the treatment
of AIDS (acquired immune deficiency syndrome), as
has 2′-3′-dideoxyinosine (DDI)
•Propose a reason for the effectiveness of these two
compounds
•Hint: How might these two compounds fit into a DNA
chain?
Example 10.1 -DNA Structure
•A nucleoside derivative that has been very much in
the news is 3′-azido-3′-deoxythymidine (AZT), as
shown in Figure 10.7
•This compound has been widely used in the treatment
of AIDS (acquired immune deficiency syndrome), as
has 2′-3′-dideoxyinosine (DDI)
•Propose a reason for the effectiveness of these two
compounds
•Hint: How might these two compounds fit into a DNA
chain?
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.7 -Structure of AZT and DDI
Figure 10.7 -Structure of AZT and DDI
© 2018 Cengage Learning. All Rights Reserved.
Example 10.1 -Solution
•Both compounds lack a hydroxyl group at the 3′-
position of the sugar moiety
•They cannot form the phosphodiester linkages found in
nucleic acids
•Thus, they interfere with the replication of the AIDS
virus by preventing nucleic acid synthesis
Example 10.1 -Solution
•Both compounds lack a hydroxyl group at the 3′-
position of the sugar moiety
•They cannot form the phosphodiester linkages found in
nucleic acids
•Thus, they interfere with the replication of the AIDS
virus by preventing nucleic acid synthesis
© 2018 Cengage Learning. All Rights Reserved.
DNA Polymerases
•Enzymes that form DNA from deoxyribonucleotides
on a DNA template
•In E. coli, there are at least five DNA polymerases,
three of which have been extensively studied
•Polymerase I (Pol I) - Consists of a single polypeptide
chain
•Polymerase II and III (Pol II and Pol III) - Multisubunit
proteins that share some common subunits
•Important considerations
•Speed of synthetic reaction, or turnover number
•Processivity: Number of nucleotides incorporated
before the dissociation of enzyme from the template
DNA Polymerases
•Enzymes that form DNA from deoxyribonucleotides
on a DNA template
•In E. coli, there are at least five DNA polymerases,
three of which have been extensively studied
•Polymerase I (Pol I) - Consists of a single polypeptide
chain
•Polymerase II and III (Pol II and Pol III) - Multisubunit
proteins that share some common subunits
•Important considerations
•Speed of synthetic reaction, or turnover number
•Processivity: Number of nucleotides incorporated
before the dissociation of enzyme from the template
© 2018 Cengage Learning. All Rights Reserved.
Table 10.1 - Properties of DNA Polymerases of
E. Coli
Table 10.1 - Properties of DNA Polymerases of
E. Coli
© 2018 Cengage Learning. All Rights Reserved.
DNA Polymerase Reactions
•DNA polymerase cannot catalyze de novo synthesis
•Require:
•Presence of a primer
•Primer: Short stretch of RNA hydrogen-bonded to the
template DNA to which the growing DNA strand is
bonded at the start of synthesis
•All four deoxyribonucleoside triphosphates
•dTTP, dATP, dGTP, and dCTP
•Mg
2+
•DNA template
•All four ribonucleoside triphosphates
•ATP, UTP, GTP, and CTP
DNA Polymerase Reactions
•DNA polymerase cannot catalyze de novo synthesis
•Require:
•Presence of a primer
•Primer: Short stretch of RNA hydrogen-bonded to the
template DNA to which the growing DNA strand is
bonded at the start of synthesis
•All four deoxyribonucleoside triphosphates
•dTTP, dATP, dGTP, and dCTP
•Mg
2+
•DNA template
•All four ribonucleoside triphosphates
•ATP, UTP, GTP, and CTP
© 2018 Cengage Learning. All Rights Reserved.
Function of DNA Polymerases
•DNA Pol I
•Repairing and patching DNA
•DNA Pol III
•Polymerization of the newly formed DNA strand
•DNA Pol II, IV, and V
•Repairing enzymes
•Exonuclease activities are part of proofreading-and-
repair functions
•Proofreading: Removing incorrect nucleotides during
DNA replication
•Repair: Removing incorrect nucleotides from DNA and
replacing them with correct ones
Function of DNA Polymerases
•DNA Pol I
•Repairing and patching DNA
•DNA Pol III
•Polymerization of the newly formed DNA strand
•DNA Pol II, IV, and V
•Repairing enzymes
•Exonuclease activities are part of proofreading-and-
repair functions
•Proofreading: Removing incorrect nucleotides during
DNA replication
•Repair: Removing incorrect nucleotides from DNA and
replacing them with correct ones
© 2018 Cengage Learning. All Rights Reserved.
Proteins Required for DNA Replication: Supercoiling
and Replication
•DNA replication is carried out by replisomes
•Replisomes: Complex of DNA polymerase, the RNA
primer, primase, and helicase at the replication fork
•DNA gyrase: Class II topoisomerase
•Catalyzes reactions involving relaxed, circular DNA
with a nick in one strand to the supercoiled form with
the nick sealed
•Energy required for the process is supplied by hydrolysis
of ATP
Proteins Required for DNA Replication: Supercoiling
and Replication
•DNA replication is carried out by replisomes
•Replisomes: Complex of DNA polymerase, the RNA
primer, primase, and helicase at the replication fork
•DNA gyrase: Class II topoisomerase
•Catalyzes reactions involving relaxed, circular DNA
with a nick in one strand to the supercoiled form with
the nick sealed
•Energy required for the process is supplied by hydrolysis
of ATP
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.9 - DNA Gyrase Introduces Supertwisting
in Circular DNA
Figure 10.9 - DNA Gyrase Introduces Supertwisting
in Circular DNA
© 2018 Cengage Learning. All Rights Reserved.
Replication with Supercoiled DNA
•Prokaryotic DNA is negatively supercoiled
•Opening the helix during replication introduces positive
supercoils ahead of the replication fork
•DNA gyrase fights positive supercoils
•Places negative supercoils ahead of the replication
fork
•Creates a swivel point at the site of the nick
•Opens and reseals the swivel point in advance of the
replication fork
•Ensures that the newly synthesized DNA automatically
assumes the supercoiled shape
•Aided by the absence of the nick at the swivel point
Replication with Supercoiled DNA
•Prokaryotic DNA is negatively supercoiled
•Opening the helix during replication introduces positive
supercoils ahead of the replication fork
•DNA gyrase fights positive supercoils
•Places negative supercoils ahead of the replication
fork
•Creates a swivel point at the site of the nick
•Opens and reseals the swivel point in advance of the
replication fork
•Ensures that the newly synthesized DNA automatically
assumes the supercoiled shape
•Aided by the absence of the nick at the swivel point
© 2018 Cengage Learning. All Rights Reserved.
Replication with Supercoiled DNA (continued)
•Helicase
•Helix-destabilizing protein
•Promotes unwinding by binding at the replication fork
•Single-strand binding protein (SSB)
•Stabilizes single-stranded regions by binding tightly to
them
Replication with Supercoiled DNA (continued)
•Helicase
•Helix-destabilizing protein
•Promotes unwinding by binding at the replication fork
•Single-strand binding protein (SSB)
•Stabilizes single-stranded regions by binding tightly to
them
© 2018 Cengage Learning. All Rights Reserved.
Primase Reaction
•RNA serves as a primer in DNA replication
•Primer activity was first observed in vivo
•Primase: Enzyme that makes a short section of RNA
to act as a primer for DNA synthesis
•Primosome: Complex at the replication fork that
consists of the RNA primer, primase, and helicase
Primase Reaction
•RNA serves as a primer in DNA replication
•Primer activity was first observed in vivo
•Primase: Enzyme that makes a short section of RNA
to act as a primer for DNA synthesis
•Primosome: Complex at the replication fork that
consists of the RNA primer, primase, and helicase
© 2018 Cengage Learning. All Rights Reserved.
Synthesis and Linking of New DNA Strands
•DNA polymerase III commences synthesis
•Newly formed DNA is linked to the 3′-hydroxyl of the
RNA primer
•Synthesis proceeds from the 5′ end to the 3′ end on
the leading and lagging strands
•As the replication fork moves away, the RNA primer
is removed by DNA Pol I and then replaced by
deoxynucleotides and DNA Pol I
•DNA ligase is responsible for the final linking of the
new strand
Synthesis and Linking of New DNA Strands
•DNA polymerase III commences synthesis
•Newly formed DNA is linked to the 3′-hydroxyl of the
RNA primer
•Synthesis proceeds from the 5′ end to the 3′ end on
the leading and lagging strands
•As the replication fork moves away, the RNA primer
is removed by DNA Pol I and then replaced by
deoxynucleotides and DNA Pol I
•DNA ligase is responsible for the final linking of the
new strand
© 2018 Cengage Learning. All Rights Reserved.
Table 10.3 -Summary of DNA Replication in
Prokaryotes
Table 10.3 -Summary of DNA Replication in
Prokaryotes
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.11 - Structural Representation of
DNA Polymerase
Figure 10.11 - Structural Representation of
DNA Polymerase
© 2018 Cengage Learning. All Rights Reserved.
Nature of Replisomes
•Studies conducted from 2010–2012 have shown that:
•Three Pol III enzymes are bound at or near the
replisome
•New Pol III is used for each Okazaki fragment
•Replisomes have one Pol III dedicated to leading
strand synthesis and two dedicated to lagging strand
synthesis
Nature of Replisomes
•Studies conducted from 2010–2012 have shown that:
•Three Pol III enzymes are bound at or near the
replisome
•New Pol III is used for each Okazaki fragment
•Replisomes have one Pol III dedicated to leading
strand synthesis and two dedicated to lagging strand
synthesis
© 2018 Cengage Learning. All Rights Reserved.
Clamp Loader
•Part of the Pol III
enzyme that opens
the sliding clamp and
inserts the DNA chain
•Pentameric enzyme
that is a member of a
family of ATPases
called the AAA+
superfamily
Clamp Loader
•Part of the Pol III
enzyme that opens
the sliding clamp and
inserts the DNA chain
•Pentameric enzyme
that is a member of a
family of ATPases
called the AAA+
superfamily
© 2018 Cengage Learning. All Rights Reserved.
Proofreading and Repair
•DNA replication takes place only once each
generation in each cell
•Mutations: Errors in replication that occur
spontaneously only once in every 10
9
–10
10
base pairs
and can be lethal to organisms
•Proofreading
•Removal of incorrect nucleotides immediately after
they are added to the growing DNA during replication
•Errors in hydrogen bonding lead to errors in a growing
DNA chain once in every 10
4
–10
5
base pairs
Proofreading and Repair
•DNA replication takes place only once each
generation in each cell
•Mutations: Errors in replication that occur
spontaneously only once in every 10
9
–10
10
base pairs
and can be lethal to organisms
•Proofreading
•Removal of incorrect nucleotides immediately after
they are added to the growing DNA during replication
•Errors in hydrogen bonding lead to errors in a growing
DNA chain once in every 10
4
–10
5
base pairs
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.13 -DNA Polymerase Proofreading
Figure 10.13 -DNA Polymerase Proofreading
© 2018 Cengage Learning. All Rights Reserved.
Nick Translation
•Cut-and-patch process catalyzed by Pol I takes place
during replication
•Cutting is the removal of the RNA primer by the 5′
exonuclease function of the polymerase
•Patching is the incorporation of the required
deoxynucleotides by the polymerase function of the
same enzyme
•Nick translation
•Removal of RNA primer or DNA mistakes by Pol I
using its 5′ → 3′ exonuclease activity as it moves along
the DNA and then filling in behind it with its
polymerase activity
Nick Translation
•Cut-and-patch process catalyzed by Pol I takes place
during replication
•Cutting is the removal of the RNA primer by the 5′
exonuclease function of the polymerase
•Patching is the incorporation of the required
deoxynucleotides by the polymerase function of the
same enzyme
•Nick translation
•Removal of RNA primer or DNA mistakes by Pol I
using its 5′ → 3′ exonuclease activity as it moves along
the DNA and then filling in behind it with its
polymerase activity
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.14 - DNA Polymerase Repair
Figure 10.14 - DNA Polymerase Repair
© 2018 Cengage Learning. All Rights Reserved.
Mutagens
•Agents that bring about a mutation
•Include:
•Ultraviolet light, which creates pyrimidine dimers
•πelectrons from two carbons on each of the two
pyrimidines form a cyclobutyl ring that distorts the
normal shape of the DNA
•Ionizing radiation
•Various chemical agents or free radicals
•Can lead to a break in the phosphodiester backbone of
the DNA strand
Mutagens
•Agents that bring about a mutation
•Include:
•Ultraviolet light, which creates pyrimidine dimers
•πelectrons from two carbons on each of the two
pyrimidines form a cyclobutyl ring that distorts the
normal shape of the DNA
•Ionizing radiation
•Various chemical agents or free radicals
•Can lead to a break in the phosphodiester backbone of
the DNA strand
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.15 - UV Irradiation Causes
Dimerization of Adjacent Thymine Bases
Figure 10.15 - UV Irradiation Causes
Dimerization of Adjacent Thymine Bases
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.16 - Oxidation Damage
Figure 10.16 - Oxidation Damage
© 2018 Cengage Learning. All Rights Reserved.
Repair Mechanisms
Mismatch repair
Base-excision
repair
Nucleotide-
excision repair
Nonhomologous
DNA end-joining
(NHEJ)
Repair Mechanisms
Mismatch repair
Base-excision
repair
Nucleotide-
excision repair
Nonhomologous
DNA end-joining
(NHEJ)
© 2018 Cengage Learning. All Rights Reserved.
Mismatch Repair
•Enzymes recognize that two bases are incorrectly
paired
•Area of mismatch is removed, and the area is
replicated again by DNA polymerases
•Challenge
•Identify which of the two strands is the correct one
•Window of opportunity arises immediately after
replication
Mismatch Repair
•Enzymes recognize that two bases are incorrectly
paired
•Area of mismatch is removed, and the area is
replicated again by DNA polymerases
•Challenge
•Identify which of the two strands is the correct one
•Window of opportunity arises immediately after
replication
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.17 -Mismatch Repair in E. Coli
Figure 10.17 -Mismatch Repair in E. Coli
© 2018 Cengage Learning. All Rights Reserved.
Base-Excision Repair
•Damaged base is removed by DNA
glycosylase leaving an AP site
(apurinic or apyrimidinic)
•Sugar and phosphate are removed
from the nucleotide by an AP
endonuclease
•Several more bases are removed by
an excision exonuclease
•DNA Pol I fills the gap, and DNA
ligase seals the phosphodiester
backbone
Base-Excision Repair
•Damaged base is removed by DNA
glycosylase leaving an AP site
(apurinic or apyrimidinic)
•Sugar and phosphate are removed
from the nucleotide by an AP
endonuclease
•Several more bases are removed by
an excision exonuclease
•DNA Pol I fills the gap, and DNA
ligase seals the phosphodiester
backbone
© 2018 Cengage Learning. All Rights Reserved.
Nucleotide-Excision Repair
•Common for DNA lesions caused by UV or chemical
means
•Section containing the lesion is removed by ABC
excinuclease
•DNA Pol I and DNA ligase work to fill the gap
Nucleotide-Excision Repair
•Common for DNA lesions caused by UV or chemical
means
•Section containing the lesion is removed by ABC
excinuclease
•DNA Pol I and DNA ligase work to fill the gap
© 2018 Cengage Learning. All Rights Reserved.
Double-Stranded Breaks (DSB)
•Breakage of both
strands of a DNA
molecule
•Pose a big threat to the
stability of the genome
•Repair mechanisms
include:
•Nonhomologous DNA
end-joining (NHEJ)
•Recombination
Double-Stranded Breaks (DSB)
•Breakage of both
strands of a DNA
molecule
•Pose a big threat to the
stability of the genome
•Repair mechanisms
include:
•Nonhomologous DNA
end-joining (NHEJ)
•Recombination
© 2018 Cengage Learning. All Rights Reserved.
DNA Recombination
•Genetic recombination: Natural process in which
genetic information is rearranged to form new
associations
•Homologous recombination: Involves a reaction
between homologous sequences
•Nonhomologous recombination: Involves
combination of different nucleotide sequences
•Occurs in specific zones of the chromosome, which
are called hot spots
DNA Recombination
•Genetic recombination: Natural process in which
genetic information is rearranged to form new
associations
•Homologous recombination: Involves a reaction
between homologous sequences
•Nonhomologous recombination: Involves
combination of different nucleotide sequences
•Occurs in specific zones of the chromosome, which
are called hot spots
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.23 - Physical Exchange of
Chromosome Parts during Recombination
Figure 10.23 - Physical Exchange of
Chromosome Parts during Recombination
© 2018 Cengage Learning. All Rights Reserved.
Holliday Model
•Describes how recombination occurs by the
breakage and reunion of DNA strands so that
physical exchange of DNA parts takes place
•Steps involved
•Alignment of two homologous DNA segments
•A nick occurs at the same place on two homologous
strands
•Strand invasion -DNAs on the two strands swap
places, or crossover, at the nick
•Crossing over proceeds down each strand of DNA
•Branch migration leads to strand exchange between
the two homologous DNA pieces
Holliday Model
•Describes how recombination occurs by the
breakage and reunion of DNA strands so that
physical exchange of DNA parts takes place
•Steps involved
•Alignment of two homologous DNA segments
•A nick occurs at the same place on two homologous
strands
•Strand invasion -DNAs on the two strands swap
places, or crossover, at the nick
•Crossing over proceeds down each strand of DNA
•Branch migration leads to strand exchange between
the two homologous DNA pieces
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.24 - Holliday Model for Homologous
Recombination
Figure 10.24 - Holliday Model for Homologous
Recombination
© 2018 Cengage Learning. All Rights Reserved.
Eukaryotic DNA Replication
•More complicated because
of:
•Multiple origins of replication
•Need to control the timing to
that of cell divisions
•Involvement of more proteins
and enzymes
•Cell growth and division
are divided into phases
•M, G
1, S, and G
2
Eukaryotic DNA Replication
•More complicated because
of:
•Multiple origins of replication
•Need to control the timing to
that of cell divisions
•Involvement of more proteins
and enzymes
•Cell growth and division
are divided into phases
•M, G
1, S, and G
2
© 2018 Cengage Learning. All Rights Reserved.
Eukaryotic Replication
•Can be initiated only by chromosomes from cells that
have reached the G
1 phase
•Step one involves various proteins
•Origin recognition complex (ORC)
•Bound to the DNA throughout the cell cycle but serves
as an attachment site for several proteins that help
control replication
•Replication activator protein (RAP)
•Protein whose binding prepares for the start of DNA
replication
•Replication licensing factors (RLFs)
•Proteins that are essential for DNA replication
•Some are cytosolic
Eukaryotic Replication
•Can be initiated only by chromosomes from cells that
have reached the G
1 phase
•Step one involves various proteins
•Origin recognition complex (ORC)
•Bound to the DNA throughout the cell cycle but serves
as an attachment site for several proteins that help
control replication
•Replication activator protein (RAP)
•Protein whose binding prepares for the start of DNA
replication
•Replication licensing factors (RLFs)
•Proteins that are essential for DNA replication
•Some are cytosolic
© 2018 Cengage Learning. All Rights Reserved.
Eukaryotic Replication (continued 1)
•Pre-replication complex (pre-RC): Combination of
the DNA, ORC, RAP, and RLFs that makes DNA
competent for replication
•Step two involves other proteins and protein kinases
•Cyclins: Proteins that are produced in one part of the
cell cycle and degraded in another
•Combine with cyclin-dependent protein kinases
(CDKs)
•Activation of cyclin–CDKs serves to initiate DNA
replication and to prevent formation of another pre-RC
Eukaryotic Replication (continued 1)
•Pre-replication complex (pre-RC): Combination of
the DNA, ORC, RAP, and RLFs that makes DNA
competent for replication
•Step two involves other proteins and protein kinases
•Cyclins: Proteins that are produced in one part of the
cell cycle and degraded in another
•Combine with cyclin-dependent protein kinases
(CDKs)
•Activation of cyclin–CDKs serves to initiate DNA
replication and to prevent formation of another pre-RC
© 2018 Cengage Learning. All Rights Reserved.
Eukaryotic Replication (continued 2)
•In the G
2 phase, DNA has been replicated
•During mitosis:
•DNA is separated into the daughter cells
•Dissolved nuclear membrane permits entrance of
RLFs that are produced in the cytosol
•Ensures that each daughter cell can initiate a new round
of replication
Eukaryotic Replication (continued 2)
•In the G
2 phase, DNA has been replicated
•During mitosis:
•DNA is separated into the daughter cells
•Dissolved nuclear membrane permits entrance of
RLFs that are produced in the cytosol
•Ensures that each daughter cell can initiate a new round
of replication
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.27 -Model for Initiation of the DNA
Replication Cycle in Eukaryotes
Figure 10.27 -Model for Initiation of the DNA
Replication Cycle in Eukaryotes
© 2018 Cengage Learning. All Rights Reserved.
Eukaryotic DNA Polymerase
•At least 19 different polymerases are present in
eukaryotes, five of which have been studied more
extensively
Eukaryotic DNA Polymerase
•At least 19 different polymerases are present in
eukaryotes, five of which have been studied more
extensively
© 2018 Cengage Learning. All Rights Reserved.
Functions of Eukaryotic DNA Polymerases
•Polymerase α-Makes primers
•Polymerase δ-Principal DNA polymerase in
eukaryotes
•Interacts with PCNA (proliferating cell nuclear antigen)
•Polymerase ε-Involved in leading strand replication
•May replace polymerase δin lagging strand synthesis
•Polymerase β-A repair enzyme
•Polymerase γ-Carries out DNA replication in
mitochondria
Functions of Eukaryotic DNA Polymerases
•Polymerase α-Makes primers
•Polymerase δ-Principal DNA polymerase in
eukaryotes
•Interacts with PCNA (proliferating cell nuclear antigen)
•Polymerase ε-Involved in leading strand replication
•May replace polymerase δin lagging strand synthesis
•Polymerase β-A repair enzyme
•Polymerase γ-Carries out DNA replication in
mitochondria
© 2018 Cengage Learning. All Rights Reserved.
PCNA
•Eukaryotic equivalent of the part of Pol III that
functions as a sliding clamp (β)
•Trimer of three identical proteins that surround the
DNA
PCNA
•Eukaryotic equivalent of the part of Pol III that
functions as a sliding clamp (β)
•Trimer of three identical proteins that surround the
DNA
© 2018 Cengage Learning. All Rights Reserved.
Table 10.5 -Differences in DNA Replication in
Prokaryotes and Eukaryotes
Table 10.5 -Differences in DNA Replication in
Prokaryotes and Eukaryotes
© 2018 Cengage Learning. All Rights Reserved.
Figure 10.29 -Basics of the Eukaryotic
Replication Fork
Figure 10.29 -Basics of the Eukaryotic
Replication Fork
© 2018 Cengage Learning. All Rights Reserved.
Biochemical Connection: Telomerase and
Cancer
•Replication of linear DNA molecules poses particular
problems at the ends of the molecules
•Telomeres - Special structures found in the ends of
eukaryotic chromosomes
•Series of repeated DNA sequences
•Noncoding and act as a buffer against degradation of
the DNA sequence at the ends
•Telomerase - Ribonuclear protein that contains a
section of RNA that is the complement of the telomere
•Provides a mechanism for synthesis of telomeres
•Reactivated in cancer cells
Biochemical Connection: Telomerase and
Cancer
•Replication of linear DNA molecules poses particular
problems at the ends of the molecules
•Telomeres - Special structures found in the ends of
eukaryotic chromosomes
•Series of repeated DNA sequences
•Noncoding and act as a buffer against degradation of
the DNA sequence at the ends
•Telomerase - Ribonuclear protein that contains a
section of RNA that is the complement of the telomere
•Provides a mechanism for synthesis of telomeres
•Reactivated in cancer cells
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