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Control of DNA replication
Replicon
Origins and terminators
Solutions to the “end problem” (telomeres)
Cellular control mechanisms
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
Replicon = unit that controls replication
InitiatorReplicator Replicator
Initiator
+
E. coli ori C DnaA
Yeast ARS ,
an autonomously
replicating sequence
ORC (the
origin recognition complex)
+ ABF1 ( ARS binding factor 1)
Complex of
Replicator +
initiator allows
replication to
begin
duplex
DNA
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
Replication “eyes”
Newly replicated strand
Parental strand
A linear molecule forms a "bubble"
when replicating.
A circular molecule forms a
"theta" when replicating.
Theta-form replication
intermediates visualized in
EM for polyoma virus
B. Hirt
Bidirectional and unidirectional replication
ori
ori
Bidirectional replication: 2 forks move in opposite directions
Unidirectional replication: A single fork moves in one direction
For completed molecules, label appears first in the fragments of DNA synthesized last
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 havethe most radioactivity.
A
C
D
E
K
FJ
GB
I
H
Physical map of the SV40 DNA fragmentsproduced by cleavage with H. influenzarestriction endonucleases
Data from labeling completed DNAs
Relative amount of pulse label
Fragment 5 min 10 min 15 minA 1.0 1.0 1.0B 3.9 3.0 2.3C 0 0.75 0.75D 0.92 0.86 1.1E 1.8 2.0 1.7F 4.0 3.1 2.4G 5.4 4.2 2.6H 1.7 2.5 2.0I 2.7 3.0 2.2J 4.9 3.7 2.6K 2.4 2.9 1.9
A
C
D
E
K
FJ
GB
I
H
Position of ori for SV40
A
C
D
E
K
F
J
G
B
I
H
ori
term
Replicating molecules have different shapes generated by replication bubbles
and forksSimple Y Bubble Double Y Asymmetric
or
Fragmentsize doublesduringreplication.
Bubble arcs on 2-D gels
Bubble
unitlength
"Bubble-arc"
A fragment containing an origin will have one or two replication forks moving through it, generating bubbles of increasing size. These willbe detected as bubble arcs on 2-D gels of the replicating DNA, when that region is used as a hybridization probe.
1st dimension: size
2nd dimension: size and SHAPE
Unit lengthTwice unit length
Y-arcs on 2-D gels of replicating molecules
2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc"Simple Y
or
Fragmentsize doublesduringreplication.
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.
1st dimension: separate by size
2nd dimension: separate by size and SHAPE
Brewer and Fangman, 1987
2-D gels: map number & position of replication origins1st dimension separates by size
2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc" "Bubble-arc" Related to distance from ori to end of fragment.
Simple Y Bubble Double Y Asymmetric
Fragmentsize doublesduringreplication.
Example of analysis of a replicon using 2-D gels
2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc"
unitlength
"Bubble-arc"2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc" 2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc" 2nd dimensionalsoseparates by shape.
unitlength
twice unitlength
"Y-arc"
Restriction fragments:
Fork movement
origin termination
Positions of oriC and ter in E. coliForks meet and terminate in this approx. 100 kb region
and are 23 bp binding sites for Tus, a "contra-helicase."terC and terB
block progress of Fork 2
terD and terAblock progress of Fork 1
E. coli chromosome
oriC
Replication fork 1
245 bp
Replication fork 2
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.
Structure of oriC
• 245 bp long– 4 copies of a 9 bp repeat– 3 copies of a 13 bp repeat– 11 GATC motifs
13 1313 9 9 99
1 GGATCCGGAT AAAACATGGT GATTGCCTCG CATAACGCGG TATGAAAATG GATTGAAGCC 61 CGGGCCGTGG ATTCTACTCA ACTTTGTCGG CTTGAGAAAG ACCTGGGATC CTGGGTATTA121 AAAAGAAGAT CTATTTATTT AGAGATCTGT TCTATTGTGA TCTCTTATTA GGATCGCACT181 GCCCTGTGGA TAACAAGGAT CCGGCTTTTA AGATCAACAA CCTGGAAAGG ATCATTAACT241 GTGAATGATC GGTGATCCTG GACCGTATAA GCTGGGATCA GAATGAGGGG TTATACACAA301 CTCAAAAACT GAACAACAGT TGTTCTTTGG ATAACTACCG GTTGATCCAA GCTTCCTGAC361 AGAGTTATCC ACAGTAGATC GCACGATCTG TATACTTATT TGAGTAAATT AACCCACGAT
Conservation of oriC in enteric bacteria
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
• 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
Proteins needed for initiation at oriC #2
• DnaG primase
• Gyrase
• SSB
• All but DnaA are also used in elongation
Proteins needed for initiation at oriC #3
Initiation at oriC: Model
Positions of ter sequences in E. coliForks meet and terminate in this approx. 100 kb region
and are 23 bp binding sites for Tus, a "contra-helicase."terC and terB
block progress of Fork 2
terD and terAblock progress of Fork 1
E. coli chromosome
oriC
Replication fork 1
245 bp
Replication fork 2
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
Termination and
resolution
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.
Regulation of replication by methylation
G A T C
C T A G
m
m
G A T C
C T A G
m
G A T C
C T A Gm
G A T C
C T A G
m
m
G A T C
C T A G
m
m
replicate methylate(lags behind replication)
Fully methylated Hemimethylated
dam methylase
Fully methylated
Will replicate Will not replicate Will replicate