1. Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 28
DNA Replication, Repair, and Recombination
Part II: DNA replication
Copyright © 2007 by W. H. Freeman and Company

3. Semi-conservative model of DNA replication
Parental strand serves as “template”

8. 2 1 3

9. Problems (because of the double helix):
Antiparallel strands (opposite strands)
Double strands
Supercoiling, unwinding

10. E. coli DNA polymerase I (Klenow fragment) Polymerase unit
3’->5’ exonuclease unit
(proofreading/correction)

11. DNA polymerases catalyze the formation of polynucleotide chains
Template-directed enzyme
(base pair-dependent)
Catalyzes nucleophilic attack by 3’-OH
on the “a” phosphate
Requires a primer with free 3’-OH
(RNA polymerase??)

12. Holds DNA
Active site
(2 metal ions)

13. participate in polymerase
Two bound metal ions
participate in polymerase
Activates 3’-OH
Stabilizes (-) charge

14. Is hydrogen bond-base pairing enough for adding the right nucleotide?

15. Fails to form H bonds but
can still direct addition of T
 1. Shape complementarity

16. major minor Residues of the enzyme form H bonds with minor
ruler
Structural study
Residues of the enzyme form H bonds with minor
groove side of the base pairs in the active site
 2. Minor groove interactions

18. Polymerases undergo conformational changes
 3. Shape selectivity

19. Where does this primer come from?
(5 nt)
Removed by hydrolysis

21. Replication fork
~1000 nt
Primer
Remove and fill in
(DNA polymerase I)
DNA ligase

22. thermodynamically
uphill rxn
nucleophilic attack
but no leaving group

23. Mechanism of DNA ligase

24. Bacterial helicase (PcrA)
ssDNA binding
ATP binding and hydrolysis

25. Separation of strands requires helicase and ATP
Both A1 and B1 bind DNA
ATP  closure, A1 releases DNA
ATP hydrolysis  open, B1 releases
Move in 3’  5’ direction

26. Helicase: large class
(5’->3’, RNA, oligomers)
Conserved residues
among helicase
ATP-induced
conformational
change
Hexameric helicase (euk)
 ATPase (AAA family)

27. DNA replication must be rapid!
Ex. E. coli:
4.6x106 bp, replicate in <40 mins  2000bp/sec
Differences in eukaryotic:
Multiple origins
Additional enzyme for telomeres
Polymerases: catalytic potency, fidelity and
processivity (catalysis without releasing substrates)
(processive vs. distributive enzymes!)

28. b2 subunit of DNA polymerase III How does DNA get in this?
Keep polymerase
associated with DNA
 Sliding DNA clamp
How does DNA get in this?
 clamp loader
(requires ATP)
35 Å

30. Topoisomerase II
(add – supercoils)
(DnaB)
(single-strand-binding)

32. DNA polymerase “holoenzyme”
Interacts with SSB
3‘5’ proofreading
DNA polymerase “holoenzyme”

33. Add 1000 nt
before releasing
& new loop
DNA pol I
Remove primers
Fill in

34. Where does replication begin??
In E. coli: a unique site “origin of replication”
is called oriC locus

35. DnaAoriC:
Preparation for replication
Bind to each others’
ATPase domains;
Breaking apart when
ATP hydrolyzed

36. Preparation for replication
DnaAoriC:
Preparation for replication
DnaB (hexameric helicase) + DnaC (helicase loader)
SSB
“Prepriming complex”
DnaG (primase) 1 2

37. DNA pol III holoenzyme + Prepriming complex ATP hydrolysis within DnaA
Breakup of DnaA
(preventing addition round of replication!) 3

38. Eukaryotic replication, why more complex?
Size of DNA (6 billion bp)
Number of chromosome (23 vs. 1)
Linear vs. circular

39. Eukaryotic replication, why more complex? Size of DNA
Number of chromosome
30,000 origins!
But no defined sequence
ORCs (origin of replication complexes) = prepriming complex
Replicon: replication unit (how many does E. coli have?)

40. Eukaryotic DNA replication
ORCsorigins
Preparation for replication
Cdc6, Cdt1  MCM2-7
(licensing factors  formation of initiation complex)
Replication protein A (=SSB)
Two distinct polymerases:
Pol a (initiator)
Pol d (replicative) 1 2 3
Polymerase switching

41. Eukaryotic DNA replication
Pol a (initiator):
Primase + polymerase (20nt)
Replication factor C (RFC):
displaces pol a
recruits PCNA (b2 of pol III)
Replication until replicons meet
(Primers, ligase) 3 4 5

43. !!!
Topo I or 2?

44. Bi-directional DNA synthesis

47. Cell cycle
Cyclins
Cyclin-dependent
protein kinase (CDK)

48. Eukaryotic replication, why more complex?
Size of DNA (6 billion bp)
Number of chromosome (23 vs. 1)
Linear vs. circular

49. !!!
Chromosome shortening
after each round of
DNA replication

50. Chromosome ends = telomeres
Telomere-binding
protein
(AGGGTT)n
Loop for
protection

51. Telomeres are replicated
by telomerase
Telomerase has:
RNA template
Reverse transcriptase

52. High levels of telomerase
in dividing cells
tumor and aging

53. Summary: DNA replication
Mode
Enzymology
Polymerization steps
Eukaryotes vs. prokaryotes
Telomeres