1. Section E DNA Replication
Molecular Biology Course
Section E
DNA Replication

2. E1: DNA Replication: An Overview
Molecular Biology Course
E1: DNA Replication: An Overview
Replicons, semi-conservative, semi-discontinous, RNA priming
E2: Bacterial DNA replication
Experimental system, initiation, unwinding, elongation, termination & segregation
E3: Eukaryotic DNA replication
Experimental system, cell cycle, initiation, replication forks, nuclear matrix, telomere repl.

3. E1: DNA Replication: An Overview
Replicons
semi-conservative mechanism
semi-discontinous replication
RNA priming

4. DNA replication
E1-1 Replicons
Replicon is any piece of DNA which replicates as a single unit. It contains an origin and sometimes a terminus
Origin is the DNA sequence where a replicon initiates its replication.
Terminus is the DNA sequence where a replicon usually stops its replication

5. Prokaryotic genome: a single circular DNA = a single replicon
DNA replication
Prokaryotic genome: a single circular DNA = a single replicon
Eukaryotic genome: multiple linear chromosomes & multiple replicons on each chromosome

6. Bidirectional replication of a circular bacterial replicon
DNA replication
Bidirectional replication of a circular bacterial replicon
All prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circlular and comprise single replicons.
There is a single termination site roughly 180o opposite the unique origin.

7. DNA replication
Linear viral DNA molecules usually have a single origin, replication details (see Section R)
In all the cases, the origin is a complex region where the initiation of DNA replication and the control of the growth cycle of the organism are regulated and co-ordinated.

8. Multiple eukaryotic replicons and replication bubbles
DNA replication
Multiple eukaryotic replicons and replication bubbles
The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple regions, each with its own orgin.
A typical mammalian cell has replicons with a size range of kb. When replication forks from adjacent replication bubbles meet, they fuse to form the completely replicated DNA. No distinct termini are required

9. replication bubbles  replication fork
DNA replication
replication bubbles  replication fork
See Page 74 of your text book

10. E1-2 Replication is Semi-conservative
DNA replication
E1-2 Replication is Semi-conservative

11. 15N labeling experiment Semi-conservative mechanism
DNA replication
Semi-conservative mechanism
15N labeled DNA
unlabeled DNA
15N labeling experiment
15N labeling: grow cells in ??
Collect DNA: grow cells in ??
Separation: method ??
Result interpretation

13. E1-3 Replication is Semi-discontinuous
DNA replication
E1-3 Replication is Semi-discontinuous

14. Semi-discontinuous replication
DNA replication
Semi-discontinuous replication
Ligation
Okazaki fragments

15. Discovery of Okazaki fragments
DNA replication
Discovery of Okazaki fragments
Evidence for semi-discontinuous replication
[3H] thymidine pulse-chase labeling experiment
Grow E. coli
Add [3H] thymidine in the medium for a few second spin down and break the cell to stop labeling  analyze  found a large fraction of nascent DNA ( nt) = Okazaki fragments
Grow the cell in regular medium then  analyze  the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments

16. DNA replication
E1-4 RNA priming
The first few nucleotides at the 5’-end of Okazaki fragments are ribonucleotides. Hence, DNA synthesis is primed by RNA that is then removed before fragments are joined. Crucial for high fidelity of replication

17. E2: Bacterial DNA replication
Experimental system
initiation,
unwinding,
elongation,
termination & segregation

18. E2-1: In vitro experimental systems
DNA replication
E2-1: In vitro experimental systems
Purified DNA: smaller and simpler bacteriophage and plasmid DNA molecules (fX174, 5 Kb)
All the proteins and other factors for its complete replications
In vitro system: Put DNA and protein together to ask for replication question

19. DNA replication
E2-2: Initiation
Study system: the E. coli origin locus oriC is cloned into plasmids to produce more easily studied minichromosomes which behave like E. coli chromosome.

20. oriC contains four 9 bp binding sites for the initiator protein DnaA
oriC contains four 9 bp binding sites for the initiator protein DnaA. Synthesis of DnaA is coupled to growth rate so that initiation of replication is also coupled to growth rate.
DnaA forms a complex of molecules, facilitating melting of three 13 bp AT-rich repeat sequence for DnaB binding.
DnaB is a helicase that use the energy of DNA hydrolysis to further melt the double-stranded DNA .
Ssb (single-stranded binding protein) coats the unwinded DNA.
DNA primase load to synthesizes a short RNA primer for synthesis of the leading strand.
Primosome: DnaB helicase and DNA primase

21. Initiation

22. Re-initiation of bacterial replication at new origins before completion of the first round of replication

23. Resolved by a type II topoisomerase called DNA gyrase
DNA replication
E2-3: Unwinding
Positive supercoiling: caused by removal of helical turns at the replication fork.
Resolved by a type II topoisomerase called DNA gyrase

24. DNA replication
E2-4: Elongation

25. DNA polymerase III holoenzyme:
a dimer complex, one half synthesizing the leading strand and the other lagging strand.
Having two polymerases in a single complex ensures that both strands are synthesized at the same rate
Both polymerases contain an a-subunit---polymerase
e-subunit---3’5’ proofreading exonuclease
b-subunit---clamp the polymerase to DNA
other subunits are different.
Replisome: in vivo, DNA polymerase holoenzyme dimer, primosome (helicase) are physically associated in a large complex to synthesize DNA at a rate of 900 bp/sec.

27. Other two enzymes during Elongation
1. Removal of RNA primer, and gap filling with DNA pol I
2. Ligation of Okazaki fragments are linked by DNA ligase.

28. Elongation: lagging strand replication
Polymerase III holoenzyme
(DNA pol III)
DNA pol I (5’3’ exonulclease activity)
DNA pol I (5’3’ polymerase activity)
DNA ligase

29. E2-5: Termination and Segregation
DNA replication
E2-5: Termination and Segregation

30. Termination
Terminus: containing several terminator sites (ter) approximately 180o opposite oirC.
Tus protein: ter binding protein, an inhibitor of the DnaB helicase
ter

31. Segregation
Topoisomerase IV: a type II DNA topoisomerase, function to unlink the interlinked daughter genomes.

32. E3: Eukaryotic DNA replication
Experimental system
cell cycle,
initiation,
replication forks,
nuclear matrix,
telomere replication.

33. E3-1: In vitro experimental systems
DNA replication
E3-1: In vitro experimental systems
Purified DNA :
All the proteins and other factors for its complete replications

34. Small animal viruses (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation.
2. Yeast (Saccharomyces cerevisiae): 1.4 X 107 bp in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system
3. Cell-free extract prepared from Xenopus (frog) eggs containing high concentration of replication proteins and can support in vitro replication.

35. E3-2: Cell cycle When to replicate
DNA replication
E3-2: Cell cycle When to replicate

36. Cell cycle G1 preparing for DNA replication (cell growth) S
a short gap before mitosis M
mitosis and cell division
Entry into the S-phase:
Cyclins
Cyclin-dependent protein kinases (CDKs)
signaling

37. E3-2: Iniation of multiple replicons
DNA replication
E3-2: Iniation of multiple replicons
Timing
Order

38. Clusters of about 20-50 replicons initiate simultaneously at defined times throughout S-phase
Early S-phase: euchromatin replication
Late S-phase: heterochromatin replication
Centromeric and telomeric DNA replicate last

39. 2. Only initiate once per cell cycle
Licensing factor:
required for initiation and inactivated after use
Can only enter into nucleus when the nuclear envelope dissolves at mitosis

40. Initiation
Licensing factor

41. Initiation: origin
Yeast replication origins (ARS- autonomously replicating sequences, enables the prokaryotic plasmids to replicate in yeast).
Minimal sequence of ARS: 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box)
Additional copies of the above sequence is required for optimal efficiency.
ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication.

42. E3-3: Replication fork & elongation
DNA replication
E3-3: Replication fork & elongation
unwinding
enzymes

43. Replication fork
Unwinding DNA from parental nucleosomes before replication : 50 bp/sec, helicases and RP-A
New nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes.

44. Elongation: three different DNA polymerases are involved.
DNA pol a: contains primase activity and synthesizes RNA primers for the leading strands and each lagging strand fragments. Continues elongation with DNA but is replaced by the other two polymerases quickly.
DNA pol d: on the leading strand that replaces DNA pol a. can synthesize long DNA
DNA pol e: on the lagging strand that replaces DNA pol a. synthesized Okazaki fragments are very short (135 bp in SV40), reflecting the amount of DNA unwound from each nucleosome.

45. DNA replication
E3-4: Nuclear Matrix
A scaffold of insoluble protein fibers which acts as an organizational framework for nuclear processing, including DNA replication, transcription

46. Replication factories: all the replication enzymes, DNA associated with the replication forks in replication
BUdR labeling of DNA
Visualizing by immunoflurescence using BUdR antiboby

47. E3-3: Telomere replication
DNA replication
E3-3: Telomere replication
Solving the problem of lagging strand synthesis
-- Chromosomal ends shortening 5’ 3’
Parental DNA 3’ 5’ 3’ 5’ 5’ 3’
Daughter DNAs 5’ 3’ 3’ 5’

48. DNA replication
telomerase

49. DNA replication
Telomerase
Contains a short RNA molecule as telomeric DNA synthesis template
Telomerase activity is repressed in the somatic cells of multicellular organism, resulting in a gradual shortening of the chromosomes with each cell generation, and ultimately cell death (related to cell aging)
The unlimited proliferative capacity of many cancer cells is associated with high telomerase activity.
See movie for cancer metastasis

50. Supplemental 1
DNA polymerase control the fidelity of DNA replication
Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain
Processive DNA polymerases have 3’5’ exonuclease activity

51. Supplemental 2
Proofreading by
E. coli polymerase

52. Crystal structure of phage T7 DNA polymerase
template
Exonuclease domain