1. Welcome to Class 18
Introductory Biochemistry

2. Lecture 18: Outline and Objectives
DNA Replication
DNA polymerase
the enzymatic reaction
proofreading and accuracy
DNA synthesis
origins & initiation
the replication fork
leading & lagging strand synthesis
termination
DNA Repair
Mutations
Mechanisms
mismatch repair
base excision repair
nucleotide excision repair
direct repair

3. Replication: DNA  DNA Semiconservative model
hybrid duplex of old and new strand
Conservative model
duplex of only old or only newly synthesized DNA
Figure 1-31

4. Replication is semiconservative
Meselson-Stahl
Experiment:
Figure 25-2
Conclusion: DNA synthesis is semi-conservative

5. DNA synthesis is performed by DNA polymerases
Requires:
template strand to copy
primer strand with 3’ OH
dNTP substrates
Catalyzes:
nucleophilic attack by 3’ OH
phosphodiester bond formation
5’→3’ synthesis

6. DNA synthesis is always 5’ → 3’
basepairing directs choice of dNTP
Primer strand 5'GTCA
Template strand 3'CAGTCAG
Figure 25-5

7. Accuracy or fidelity in replication
Base pair geometry
is important
Figure 25-6

8. All DNA polymerases have a 3’→5’ exonuclease activity
Which polymerase replicates the genome?
Pol I is the only polymerase with a 5’→3’ exonuclease activity
All DNA polymerases have a 3’→5’ exonuclease activity

9. Accuracy is essential 3’ → 5’ exonuclease activity is for proofreading
DNA polymerases insert one incorrect nucleotide for every 104 to 105 correct ones.
Proofreading improves the inherent accuracy of the polymerization reaction by 100- to 1000-fold.
In combination, one net error for every 106 to 108 bases added.
Figure 25-7

10. DNA Replication has Three Major Stages
Initiation
Elongation
Termination
Figure 25-3
Replication Initiates at Origins

11. Relication of a circular chromosome
Check this one could be figure 25-3 but it is not the same info being displayed
Tritium labeling experiments show that both strands
are replicated at the same time.
Figure 25-3

12. The E. Coli chromosome
The origin
Figure 25-1

13. Initiation of replication requires specific sequences and proteins
DUE = DNA unwinding element (contains high amount AT)
Figure 25-10

14. Model for initiation of replication
Figure 25-11

15. Elongation: Priming RNA primers primase 5’ 3’ 3’ 5’ Origin 5’ 5’ 3’ 3’

16. Elongation: Polymerization5’ 5’ 3’ 3’ 5’ 5’
Origin
DNA polymerase III
DNA Polymerases Synthesize Only 5’→3’ 5’
How are the other strands copied? 5’ 3’ 3’ 5’ 5’
Origin

17. Elongation: Polymerization5’ 5’ 3’ 3’ 5’ 5’
Leading Strands
Origin 5’ 5’ 5’ 3’ 3’ 5’ 5’ 5’
Lagging Strands
Origin

18. Elongation: DNA replication is semidiscontinuous
Leading strand synthesis
is continuous
(and in the direction of fork movement)
Lagging strand synthesis
is discontinuous
(and opposite to fork movement)
Figure 25-4

19. Elongation: Lagging strand synthesis
Figure 25-12

20. Elongation: Removal of RNA primers
5’→3’ exo activity
5’→3’ polymerase activity
Figure 25-15

21. Elongation: Removal of RNA primers by Pol I
5’→3’ exo activity
removes RNA
5’→3’ polymerase activity
fills in with DNA
RNA replaced with DNA
Figure 25-8

22. DNA ligase: sealing the nick
Phosphodiester bond formation
adenylylation of enzyme
activation of 5’ phosphate
nucleophilic attack by 3’ OH
Figure 25-16

23. Steps in elongation Leading Strand Both Lagging Strand
 For the leading strand-
The primosome synthesizes
an RNA primer at the origin
 For the lagging strand-
The primosome synthesizes
an RNA primer for each
Okazaki fragment
 dNTPs are added by
DNA Polymerase III
 as the replication fork moves,
DnaB helicase unwinds the DNA
SSB stabilizes the single strands
DNA gyrase relieves the strain caused by unwinding
 RNA primers are removed by DNA polymerase I and the nicks are closed by DNA ligase

24. Elongation: Overview
Figure 25-12

25. The DNA pol III enzyme Polymerase activity Polymerase activity
Increases processivity
Figure (5th edition)

26. The DNA pol III clamp loader
Figure 25-14

27. DNA synthesis on the leading and lagging strands
Figure 25-13

28. DNA Replication has Three Major Stages
Initiation
Elongation
Termination
Figure 25-3
Replication initiates at origins

29. Termination of replication
Replication forks stop at the terminus region
Figure 25-17

30. Termination: the final stages
Figure 25-18

31. Replication in eukaryotes is both similar and more complex
E. coli Humans
chromosome(s) circular linear
length mm 100 mm (avg.)
replication rate ~50 nt/sec ~5 nt/sec
The replication rate is slower, and the chromosomes are longer - -
How does this work?

32. Eukaryotic chromosomes are long and linear
multiple origins of replication
Multiple origins of replication are necessary to replicate large chromosomes
Chromosomes must be replicated only once per cell cycle

33. Initiation of eukaryotic DNA replication requires two steps
(helicase)
2. Coordinate Activation
1. Formation of the Pre-RC
CDK enzymes off
CDK enzymes on
pre-RC: pre-replicative complex
Figure 25-19

34. Eukaryotic chromosomes are long and linear
multiple origins of replication
linear chromosomes present a problem...

35. The ends of linear chromosomes present a replication problem3’ 5’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’
Maybe figure 25-30 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3' 3’ 5’
Not replicated!

36. Eukaryotic chromosomes are long and linear
multiple origins of replication
Telomeres are repeated sequences
e.g., 5’- (TxGy)n (x, y = 14)
3’- (AxCy)n
which stabilize the ends of linear chromosomes
linear chromosomes present a problem...

37. Telomerase adds telomeres to chromosome ends
Telomerase synthesizes DNA from an RNA template
(a reverse transcriptase)
The template is an RNA molecule that is part of the enzyme
Telomerase is an RNP enzyme
Figure 26-38

38. Reverse transcription: RNA-dependent DNA synthesis
Figure 26-31

39. DNA repair Mutation: a permanent change in the DNA sequence
Mutations can be:
silent → no effect on gene function
deleterious → impairs gene function
advantageous → enhances gene function
Mutations can lead to:
genetic diversity
cancer in somatic cells
birth defects in germ cells

40. Mutations
Can be caused by:
Mistakes in replication
DNA Damage

41. Deamination A good reason for having T instead of U in DNA
Spontaneously,
~ 100/day
Deaminating agents
induce these conversions
at high levels
Spontaneously,
~ 1/day
Figure 8-30a

42. Deaminating agents
metabolized to Nitrous Acid (HNO2), a strong deaminating agent
Figure 8-32a

43. Depurination can occur: spontaneously,
through the action of alkylating agents
N7 alkylation increases depurination
hydrolysis
Figure 8-30

44. UV irradiation is another source of DNA damage
Generates a block to replication
Defects in repair of this lesion lead to Xeroderma pigmentosum
Figure 8-31

45. Alkylating agents
Figure 8-32b

46. Alkylation can change base-pairing properties
Figure 25-27a
cannot pair with C

47. DNA damage can result in mutations
DNA damage on one strand can be repaired using information from the other strand
Mistake is
replicated
Mutation!
Figure 25-27b

48. DNA Repair Is necessary to repair DNA damage Four Major Mechanisms:
1. Mismatch Repair
2. Base Excision Repair
3. Nucleotide Excision Repair
4. Direct Repair

49. Mismatch repair allows correction of replication errors
parental strand
is marked
a window
of opportunity
N6 methyl-A
still pairs with T
Figure 25-21
Methylation distinguishes
between template and
newly synthesized strands

50. Mismatch repair mismatch MutL-MutS binds to mismatch MutL-MutS + MutH
“finds” Me site
The methylated site
could be 1,000 bp
from the mismatch
site (either 5’ or 3’).
MutH cleaves
unmodified strand
Figure 25-22

51. Mismatch repair
Exonuclease activity (5’→3’ or 3’→5’) degrades DNA from Me past mismatch
DNA Polymerase III replaces DNA
(copies methylated strand)
End Result:
DNA containing mismatch is resynthesized
Figure 25-23

52. Base excision repair cleaves N-glycosyl bond removes sugar
damaged base
cleaves
N-glycosyl
bond
removes
sugar
nick is sealed
Different glycosylase for each base lesion
Figure 25-24

53. Nucleotide-excision repair
Excinuclease:
excision endonuclease
makes 2 cuts
excises the damaged DNA
Used for removal of large bulky lesions (i.e. pyrimidine dimers)
Figure 25-25

54. Direct repair does not remove base or nucleotide
Repairs the defect directly BUT it’s expensive
"Suicide Enzyme"
Cost = one protein inactivated per repair
p. 1033

55. Direct repair of alkylated bases by AlkB in E. coli
Oxidative demethylation by an a-ketoglutarate-iron dependent dioxygenase
Figure 25-28

56. Information Pathways
Coming up:
transcription!