Replication_Basic_L4.ppt. Ucc7figgivh7ubh

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1. Outline for ReplicationReplication section of BIO 319 Fall 2007
Chapter 11
Problems: C8, C16, C24, C28, C30
I. General Features of Replication
A. Semi-Conservative
B. Starts at Origin
C. Bidirectional
D. Semi-Discontinuous
II. Identifying Proteins and Enzymes of Replication
III. Detailed Examination of the Mechanism of Replication
A. Initiation
B. Priming
C. Elongation
D. Proofreading and Termination

2. Figure 11.1Figure 11.1
Identical
base
sequences
5’
5’
3’
3’ 5’
5’
3’
3’
Watson/Crick proposed mechanism of DNA replication

3. •In the late 1950s, three different mechanisms
were proposed for the replication of DNA
–Conservative model
•Both parental strands stay together after DNA replication
–Semiconservative model
•The double-stranded DNA contains one parental and one
daughter strand following replication
–Dispersive model
•Parental and daughter DNA are interspersed in both strands
following replication
Proposed Models of DNA ReplicationProposed Models of DNA Replication

4. Figure 11.2 Three models for DNA replication

5. •Matthew Meselson and Franklin Stahl
experiment in 1958
–Grow E. coli in the presence of
15
N (a heavy isotope of
Nitrogen) for many generations
•Cells get heavy-labeled DNA
–Switch to medium containing only
14
N (a light isotope of
Nitrogen)
–Collect sample of cells after various times
–Analyze the density of the DNA by centrifugation using
a CsCl gradient

6. CsCl Density Gradient CentrifugationCsCl Density Gradient Centrifugation
15
N
14
N
DNA

7. 0
1
2
3
Generation
15
N
Shift cells to
14
N

8. Interpreting the Data
After one generation,
DNA is “half-
heavy”
After ~ two generations, DNA
is of two types: “light” and
“half-heavy”
This is consistent with only the
semi-conservative model

9. •DNA synthesis begins at a site termed the
origin of replicationorigin of replication
•Each bacterial chromosome has only one
•Synthesis of DNA proceeds bidirectionallybidirectionally
around the bacterial chromosome
–eventually meeting at the opposite side of the
bacterial chromosome
•Where replication ends
BACTERIAL REPLICATION BACTERIAL REPLICATION

10. Figure 11.4 Overview of bacterial DNA replicationFigure 11.4 Overview of bacterial DNA replication

11. Autoradiography: Radioactivity darkens film
Radioactive bacterial colonies on an agar petri dish

12. Replication Starts at an Replication Starts at an OriginOrigin and is and is BidirectionalBidirectional

13. Directionality of the DNA strands at a replication forkDirectionality of the DNA strands at a replication fork
Leading strand
Lagging strand
Fork movementFork movement

14. Experimental demonstration of semi-discontinuoussemi-discontinuous Replication
3
H labeled Okazaki fragments seen in sucrose density gradients
Wild-type cells DNA Ligase deficient cells
Sucrose gradient
Top=
Smaller
Bottom=
Bigger

15. II. Identifying Proteins and Enzymes involved in ReplicationII. Identifying Proteins and Enzymes involved in Replication
A. Combine Genetics and Biochemistry
1. Genetic Approach: Obtain Mutants that are defective in Replication
a.Such mutations are Lethal!
c. Temperature sensitive (ts) lethal
b. Conditional lethal
2. Method:
a. Mutagenize cells
b. Plate the cells on agar plates and grow at 30
o
C
c. Replica plate and grow at 37
o
C

16. 3. Identifying which ts lethal mutants have defects in Replication
a. pick ts colonies from 30
o
C plate and grow them in liquid medium at 30
o
C.
b. shift them to 37
o
C
c. add Bromodeoxyyridine (BrdU) and continue growth for a short time at 37
o
C
d. remove the BrdU and irradiate the cells with UV light
1). if BrdU is incorporated into the DNA the UV light will kill the cells
e. return the cells to 30
o
C
f. the cells that revive and continue to grow did not incorporate BrdU
because they have a defect in Replication!!! Replication!!!
g. Those ts cells that had other defects continued to replicate their DNA
and incorporate BrdU, and hence the UV light killed them.

17. •The in vitro study of DNA replication was pioneered by
Arthur Kornberg
–Nobel Prize in 1959
Kornberg mixed the following
–An extract of proteins from E. coli
–Template DNA
–Radiolabeled nucleotides
•Incubated to allow the synthesis of new DNA strands
–Addition of acid will precipitate these DNA strands
–Centrifugation will separate them from the radioactive nucleotides
DNA Replication In VitroDNA Replication In Vitro

18. In vitro Complementation of mutants in DNA Replication
WT
37C
DNA
ts mutant
30C
DNA
ts mutant
37C
DNA
ts mutant WT
37C
DNA

19. •The origin of replication in E. coli is termed oriC
–origin of Chromosomal replication
•Important DNA sequences in oriC
–AT-rich regionAT-rich region
–DnaA boxesDnaA boxes
Initiation of Replication

20. Figure 11.5 DNA sequences at the Bacterial origin of Replication Figure 11.5 DNA sequences at the Bacterial origin of Replication

21. FigureFigure 11.6 Initiation of Replication at oriC11.6 Initiation of Replication at oriC
•DNA replication is initiated by the binding of
DnaA proteins to the DnaA box sequences
–causes the region to wrap
around the DnaA proteins and
separates the AT-rich region

22. Figure 11.6 continued
Uses energy from ATP to
unwind the duplex DNA
SSB
SSB
SSB
SSB

23. •DNA helicase separates the two DNA strands by
breaking the hydrogen bonds between them
•This generates positive supercoiling ahead of
each replication fork
–DNA gyrase travels ahead of the helicase and alleviates
these supercoils
•Single-strand binding proteins bind to the
separated DNA strands to keep them apart
•Then short (10 to 12 nucleotides) RNA primers are
synthesized by DNA primase
–These short RNA strands start, or prime, DNA synthesis

24. Fig. 11.9a(TE Art)
Able to
covalently link
together
Unable to
covalently link
the 2 individual
nucleotides together
P
rim
e
r
5’
5’
5’
5’
5’
5’
3’
3’
3’
3’
3’
DNA Polymerase Cannot Initiate new Strands

25. 11-30
Figure 11.10
Innermost
phosphate

26. DNA Polymerase III- does the bulk of copying DNA in Replication

27. Figure 11.8 Schematic representation of DNA Polymerase IIIFigure 11.8 Schematic representation of DNA Polymerase III
Structure resembles a
human right hand
Template DNA thread
through the palm;
Thumb and fingers
wrapped around the DNA

28. Figure 11.7 Two dimensional view of a replication forkFigure 11.7 Two dimensional view of a replication fork
D
ir
e
c
t
io
n
o
f
s
y
n
th
e
s
is
o
n
la
g
g
in
g
s
t
r
a
n
d
Direction of synthesis
on leading strand
3’
5’
3’
5’
3’
5’

29. Figure 11.13 “Three Dimensional” view of Replication Fork
Direction of fork movement
Direction of synthesis
Of lagging strand
Direction of synthesis
of leading strand

30. Proofreading by the 3’  5’ exonuclease activity of DNA
polymerases during DNA replication.

31. Nicks Nicks are single strand breaks in double stranded DNA
KnicksKnicks are something else all together!

32. Synthesis and replacement of RNA primers during DNA replicationSynthesis and replacement of RNA primers during DNA replication

33. •DNA polymerases can only synthesize DNA only in the 5’ to
3’ direction and cannot initiate DNA synthesis
•These two features pose a problem at the 3’ end of linear
chromosomes
Figure 11.24 Problem at ends of eukaryotic linear ChromosomesFigure 11.24 Problem at ends of eukaryotic linear Chromosomes

34. •If this problem is not solved
–The linear chromosome becomes progressively shorter
with each round of DNA replication
•The cell solves this problem by adding DNA
sequences to the ends of chromosome: telomerestelomeres
–Small repeated sequences (100-1000’s)
•Catalyzed by the enzyme telomerasetelomerase
•Telomerase contains protein and RNA
–The RNA functions as the template
–complementary to the DNA sequence found in the
telomeric repeat
•This allows the telomerase to bind to the 3’ overhang

35. 11-80
Figure 11.25Figure 11.25
Step 1 = Binding
Step 3 = Translocation
The binding-
polymerization-
translocation cycle can
occurs many times
This greatly lengthens
one of the strands
The complementary
strand is made by primase,
DNA polymerase and ligase
RNA primer
Step 2 = Polymerization

36. Viral Lifecycle of a Retrovirus (HIV)Viral Lifecycle of a Retrovirus (HIV)

37. Brand
Na
me
Generic Name Abbreviation
Experimental
Code
Pharmaceutical Company
Atripla™
tenofovir DF + emtricitabine +
efavirenz*
TDF + FTC + EFV 
Bristol-Myers Squibb &
Gilead Sciences
Combivir
®
zidovudine + lamivudine AZT + 3TC  GlaxoSmithKline
Emtriva®emtricitabine FTC  Gilead Sciences
Epivir® lamivudine 3TC   GlaxoSmithKline
Epzicom
™
abacavir + lamivudine ABC + 3TC  GlaxoSmithKline
Hivid® zalcitabine ddC  Hoffmann-La Roche
Retrovir®zidovudine AZT or ZDV  GlaxoSmithKline
Trizivir®
abacavir + zidovudine +
lamivudine
ABC + AZT +
3TC
  GlaxoSmithKline
Truvada®tenofovir DF + emtricitabineTDF + FTC   Gilead Sciences
Videx® didanosine: buffered versionsddI BMY-40900 Bristol-Myers Squibb
Videx
® E
C
didanosine: delayed-release
capsules
ddI  Bristol-Myers Squibb
Viread®
tenofovir disoproxil fumarate
(DF)
TDF or Bis(POC)
PMPA
  Gilead Sciences
Zerit® stavudine d4T BMY-27857 Bristol-Myers Squibb
Ziagen® abacavir ABC 1592U89 GlaxoSmithKline
Racivir®  RCV PSI-5004 Pharmasset
  amdoxovir AMDX or DAPD  RFS Pharma
  apricitabine APR
AVX754
(SPD754)
Avexa Limited
  elvucitabine Beta-L-Fd4C ACH-126,443 Achillion Pharmaceuticals