Control of DNA replication

1
Control of DNA replication

• Replicon
• Origins and terminators
• Solutions to the end problem (telomeres)
• Cellular control mechanisms

2
3 stages to replication

• Initiation begin at a specific site, e.g. oriC
  for E. coli.
• Elongation movement of the replication fork
• Termination at ter sites for E. coli

3
Replicon unit that controls replication
Replicator cis-acting DNA sequence required for
initiation defined genetically Origin site at
which DNA replication initiates defined
biochemically Initiator protein needed for
initiation, acts in trans
4
Replication eyes
5
Theta-form replication intermediates visualized
in EM for polyoma virus
B. Hirt
6
Bidirectional and unidirectional replication
7
For completed molecules, label appears first in
the fragments of DNA synthesized last
8
Labeling of completed DNA molecules can map
replication origins
Dana and Natahans, 1972, PNAS map the
replication origin of SV40 by labeling
replicating molecules for increasing periods of
time, isolating complete molecules, digesting
with Hind restriction endonucleases, and
determining which fragments have the most
radioactivity.
9
Data from labeling completed DNAs
Relative amount of pulse label Fragment 5
min 10 min 15 min A 1.0 1.0 1.0 B 3.9 3.0 2.3
C 0 0.75 0.75 D 0.92 0.86 1.1 E 1.8 2.0 1.7
F 4.0 3.1 2.4 G 5.4 4.2 2.6 H 1.7 2.5 2.0 I
2.7 3.0 2.2 J 4.9 3.7 2.6 K 2.4 2.9 1.9
10
Position of ori for SV40
11
Replicating molecules have different shapes
generated by replication bubbles and forks
12
Bubble arcs on 2-D gels
1st dimension size
2nd dimension size and SHAPE
Twice unit length
Unit length
A fragment containing an origin will have one or
two replication forks moving through it,
generating bubbles of increasing size. These
will be detected as bubble arcs on 2-D gels of
the replicating DNA, when that region is used as
a hybridization probe.
13
Y-arcs on 2-D gels of replicating molecules
1st dimension separate by size
2nd dimension separate by size and SHAPE
A replication fork moving through a region will
show a Y-arc on 2-D gels of the replicating DNA,
when that region is used as a hybridization probe.
Brewer and Fangman, 1987
14
2-D gels map number position of replication
origins
15
Example of analysis of a replicon using 2-D gels
16
Positions of oriC and ter in E. coli
Replication fork 2
17
Features of oriC

• oriC was identified by its ability to confer
  autonomous replication on a DNA molecule, thus it
  is a replicator.
• Studies show that chromosomal DNA synthesis
  initiates at oriC, thus it is also an origin of
  replication.
• Replication from oriC is bidirectional.

18
Structure of oriC
13
13
13
9
9
9
9

• 245 bp long
• 4 copies of a 9 bp repeat
• 3 copies of a 13 bp repeat
• 11 GATC motifs

1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG
TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA
ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA 121
AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA
TCTCTTATTA GGATCGCACT 181 GCCCTGTGGA TAACAAGGAT
CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT 241
GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA
GAATGAGGGG TTATACACAA 301 CTCAAAAACT GAACAACAGT
TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC 361
AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT
TGAGTAAATT AACCCACGAT
19
Conservation of oriC in enteric bacteria
20
Proteins needed for initiation at oriC 1

• DnaA
• Only used at initiation
• Mutations cause a slow-stop phenotype
• Binds to the 4 copies of 9 bp repeats
• Further cooperative binding brings in 20 to 40
  DnaA monomers
• Melts the DNA at the 3- 13 bp repeats

21
Proteins needed for initiation at oriC 2

• DnaB
• ATP-dependent helicase
• Displaces DnaA and unwinds DNA further to form
  replication forks
• Activates primase, apparently by stablizing a
  secondary structure in single-stranded DNA
• DnaC
• Is in complex with DnaB before loading onto
  template

22
Proteins needed for initiation at oriC 3

• DnaG primase
• Gyrase
• SSB
• All but DnaA are also used in elongation

23
Initiation at oriC Model
24
Positions of ter sequences in E. coli
Replication fork 2
25
Termination of replication DNA sites and
proteins needed

• DNA sites ter sequences, 23 bp
• terD and terA block progress of
  counter-clockwise fork, allow clockwise fork to
  pass
• terC and terB block progress of clockwise fork,
  allow counter- clockwise fork to pass
• Protein Tus
• ter utilization substance
• Binds to ter
• Prevents helicase action from a specific
  replication fork

26
Termination and resolution
27
Control by methylation

• GATC motifs are substrates for methylation by dam
  methylase.
• Methylase transfers a methyl group from
  S-adenosylmethionine to N-6 of adenine in GATC.
• Methylated GATC on BOTH strands oriC will serve
  as an origin
• Methylated GATC on ONLY one strand
  (hemimethylated) oriC is not active
• Re-methylation is slow, delays use of oriC to
  start another round of replication.

28
Regulation of replication by methylation
m
m
G A T C
G A T C
C T A G
C T A G
m
m
G A T C
methylate
replicate
C T A G
m
(lags
m
G A T C
G A T C
behind
replication)
C T A G
C T A G
m
m
dam
methylase
Hemimethylated
Fully methylated
Fully methylated
Will not replicate
Will replicate
Will replicate
29
Replicores in E. coli
30
Correlation between genome structure and
replication direction in E. coli

• Information from whole-genome sequencing
• Most genes are transcribed in the direction that
  the replication fork moves.
• GgtC on leading strand (top strand for replicore
  1, opposite strand for replicore 2)
• Oligonucleotides containing CTG are more frequent
  on the leading strand (i.e. template for lagging
  strand) in E. coli. These are the binding sites
  for DnaG (primase).
• Recombination hotspot Chi more abundant on
  leading strand