Section E - DNA Replication

1
Section E - DNA Replication
2
Contents

• E1 DNA Replication an overview
• Semi-conservative mechanism, Replicons,
  Origins and termini, Semi-discontinuous
  replication, RNA priming
• E2 Bacterial DNA replication
• Experimental systems, Initiation, Unwinding,
  Elongation, Termination and segregation
• E3 The cell cycle
• The cell cycle, Cell cycle phases,
  Checkpoints and their regulation, Cyclins and
  cyclin-dependent kinases, Regulation by E2F and
  Rb
• E4 Eukaryotic DNA replicatiom
• Experimental systems, Origins and initiation,
  Replication forks, Nuclear matrix, Telomere
  replication

3
E1 DNA Replication an overview
Semi-conservative mechanism

• A summary of the three postulated methods of DNA
  synthesis

4

• The Meselson - Stahl Experiment

5
(No Transcript)
6
E1 DNA Replication an overview
Replicons, Origins and termini

• 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.

7
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.

8

• 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.

9
Multiple eukaryotic replicons and replication
bubbles

• The long, linear DNA molecules of eukaryotic
  chromosomes consist of multiple regions, each
  with its own origin.
• A typical mammalian cell has 50000-100000
  replicons with a size range of 40-200 kb. When
  replication forks from adjacent replication
  bubbles meet, they fuse to form the completely
  replicated DNA. No distinct termini are required.

replication bubbles ? replication fork
10
E1 DNA Replication an overview
Semi-discontinuous replication

• Many enzymes are involved in the DNA replication
  fork.

11
(No Transcript)
12
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 (1000-2000 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

13
(No Transcript)
14
E1 DNA Replication an overview RNA priming

• The leading strand and all lagging strand
  fragment are primed by synthesis of a short piece
  of RNA which is then elongated with DNA. The
  primers are removed by DNA before ligation. The
  mechanism helps to maintain high replication
  fidelity.
• 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

15
E2 Bacterial DNA replication
Experimental systems

1. Purified DNA smaller and simpler bacteriophage
   and plasmid DNA molecules (fX174, 5 Kb)
2. All the proteins and other factors for its
   complete replications

In vitro system Put DNA and protein together to
ask for replication question
16
E2 Bacterial DNA replication Initiation
Re-initiation of bacterial replication at new
origins before completion of the first round of
replication
Study system the E. coli origin locus oriC is
cloned into plasmids to produce more easily
studied minichromosomes which behave like E. coli
chromosome.
17
(No Transcript)
18

1. 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.
2. DnaA forms a complex of 30-40 molecules,
   facilitating melting of three 13 bp AT-rich
   repeat sequence for DnaB binding.
3. DnaB is a helicase that use the energy of ATP
   hydrolysis to further melt the double-stranded
   DNA .
4. Ssb (single-stranded binding protein) coats the
   unwinded DNA.
5. DNA primase load to synthesizes a short RNA
   primer for synthesis of the leading strand.
6. Primosome DnaB helicase and DNA primase

19
E2 Bacterial DNA replication Unwinding

• Positive supercoiling caused by removal of
  helical turns at the replication fork.
• Resolved by a type II topoisomerase called DNA
  gyrase

20
E2 Bacterial DNA replication Elongation
21

• 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---polymeras
  e
• 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.
22
(No Transcript)
23
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.
24
E2 Bacterial DNA replication
Termination and segregation

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

25
Segregation

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

26
E3 The cell cycle The cell cycle
The cell cycle, or cell-division cycle, is the
series of events that take place in a cell
leading to its replication.
27
E3 The cell cycle Cell cycle phases
G1 preparing for DNA replication (cell
growth) S DNA replication G2 a short gap before
mitosis M mitosis and cell division
28
E3 The cell cycle Checkpoints and their
regulation

• The cell cycle is regulated in response to the
  cells environment and to avoid the proliferation
  of damaged cells.
• Checkpoint are stages at which the cell cycle may
  be halted if the circumstance are not right for
  cell division.

29
(No Transcript)
30
E3 The cell cycle Cyclins and cyclin-dependent
kinases

• The cell cycle is controlled through protein
  phosphorylation(???), which is catalysed(??) by
  multiple protein kinase complexes.
• These complexes consist of cyclins(????????), the
  regulatory subunits, and cyclin-dependent
  kinases( CDKs)

31
(No Transcript)
32
E3 The cell cycle Regulation
by E2F and Rb

• E2F family members play a major role during the
  G1/S transition in the mammalian cell cycle.
• Among E2F transcriptional targets are cyclins,
  CDKs, checkpoints regulators, DNA repair and
  replication proteins.
• The activity of E2F is inhibited by the binding
  of the protein Rb (the retinoblastoma tumor
  suppressor protein) and related proteins.

33
(No Transcript)
34
E4 Eukaryotic DNA replication
Experimental systems

1. 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
E4 Eukaryotic DNA replication
Origins and initiation

• 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

36

• 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

37
Initiation
Licensing factor
38
Initiation origin

• Yeast replication origins (ARS- autonomously
  replicating sequences, enables the prokaryotic
  plasmids to replicate in yeast).
• Minimal sequence of ARS 11 bp
  A/TTTTATA/GTTTA/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.

39
E4 Eukaryotic DNA replication
Replication forks

• The replication fork is a structure that forms
  within the nucleus during DNA replication.
• It is created by helicases(????), which break the
  hydrogen bonds holding the two DNA strands
  together.
• The resulting structure has two branching
  "prongs", each one made up of a single strand of
  DNA, that are called the leading and lagging
  strands. DNA polymerase creates new partners for
  the two strands by adding nucleotides.

40
(No Transcript)
41
E4 Eukaryotic DNA replicatiom
Nuclear matrix

• A scaffold of insoluble protein fibers which acts
  as an organizational framework for nuclear
  processing, including DNA replication,
  transcription

42
Replication factories all the replication
enzymes, DNA associated with the replication
forks in replication
BUdR labeling of DNA
Visualizing by immunoflurescence using BUdR
antiboby
43
E4 Eukaryotic DNA replication
Telomere replication
44
Telomerase

1. Contains a short RNA molecule as telomeric DNA
   synthesis template
2. 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)
3. The unlimited proliferative capacity of many
   cancer cells is associated with high telomerase
   activity.

45
DNA polymerase control the fidelity of DNA
replication
Processive DNA polymerases have 3?5 exonuclease
activity
46
Solving the problem of lagging strand synthesis
-- Chromosomal ends shortening
5
3
Parental DNA
5
3
3
5
5
3
Daughter DNAs
5
3
5
3
47
telomerase
48

• 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 which
  are very short (135 bp in SV40), reflecting the
  amount of DNA unwound from each nucleosome.

49
Crystal structure of phage T7 DNA polymerase
template
Exonuclease domain
50
Multiple choice questions

• 1.The number of replicons in a typical mammalian
  cell is .
• A 40-200.
• B 400.
• C 1000-2000.
• D 50000-100000.
• 2. In prokaryotes,the lagging strand primers are
  removed by .
• A 3' to 5' exonuclease.
• B DNA ligase.
• C DNA polymerase I.
• D DNA polymerase III.

51

• 3. The essential initiator protein at the E.
  coli origin of replication is .
• A DnaA.
• B DnaB.
• C DnaC.
• D DnaE.
• 4. Which phase would a cell enter if it was
  starved of mitogens before the R point?
• A G1.
• B S.
• C G2.
• D G0.

52

• 5. Which one of the following statements is
  true?
• A once the cell has passed the R point, cell
  division is inevitable.
• B the phosphorylation of Rb by a G1 cyclin-CDK
  complex is a critical requirement for entry into
  S phase .
• C phosphorylation of E2F by a G1 cyclin-CDK
  complex is a critical requirement for entry into
  S phase.
• D cyclin D1 and INK4 p16 are tumor suppressor
  proteins.
• 6. In eukaryotes, euchromatin replicates
  predominantly .
• A in early S-phase.
• B in mid S-phase.
• C in late S-phase.
• D in G2-phase.

53

• 7. Prokaryotic plasmids can replicate in yeast
  cells if they contain a cloned yeast .
• A ORC.
• B CDK.
• C ARS.
• D RNA.

54
THANK YOU !