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
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”
Replication Eyes
Parental strand
Newly replicated 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
Distinguishing between bidirectional and unidirectional replication
Pattern of s
EM-autorad
Bidirectional replication: 2 forks move in opposite directions
ori
ori
Unidirectional replication: A single fork moves in one direction
ori
Label newly replicating DNA first with a low specific activity nucleotide and finally with a high specific
activity nucleotide; isolate DNA, spread it on a surface and cover with a photographic emulsion. Exposure
ori
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
Physical map of the SV40 DNA fragments
increasing periods of time, isolatingproduced
complete
molecules,
digesting
by cleavage
with H.
influenza with
endonucleases
Hind restriction endonucleases, andrestriction
determining
which fragments have
the most radioactivity.
C
D
A
Physical map of the SV40 DNA fragments
produced by cleavage with H. influenza
restriction endonucleases
H
K
I
C
A
E
F
D
B
G
E
J
Data from labeling completed DNAs
Relative amount of pulse label
Fragment
A
B
C
D
E
F
G
H
I
J
K
5 min
1.0
3.9
0
0.92
1.8
4.0
5.4
1.7
2.7
4.9
2.4
10 min 15 min
1.0
1.0
3.0
2.3
0.75 0.75
0.86 1.1
2.0
1.7
3.1
2.4
4.2
2.6
2.5
2.0
3.0
2.2
3.7
2.6
2.9
1.9
Physical map of the SV40 DNA fragments
produced by cleavage with H. influenza
restriction endonucleases
C
D
A
E
H
K
I
F
B
G
J
Position of ori for SV40
ori
C
D
A
E
H
K
I
F
B
G
term
J
Replicating molecules have different
shapes generated by replication bubbles
and forks
Simple Y
Fragment
size
doubles
during
replication.
or
Bubble
Double Y
Asymmetric
Bubble arcs on 2-D gels
1st dimension separates by size
1st dimension: size
Double Y
Asymmetric
Bubble
2nd
dimension
also
separates
by shape.
"Bubble-arc"
"Y-arc"
2nd
dimension:
size and
SHAPE
twice
unit
length
Twice unit length
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.
Re
fro
fra
Y-arcs on 2-D gels of replicating molecules
dimension separates
by size
1st 1st
dimension:
separate
by size
Simple Y
Bubble
2nd
dimension
also
separates
by shape.
Fragment
size
doubles
during
replication.
or
Double Y
"Bubble-arc"
"Y-arc"
2nd
dimension:
separate by
size and
SHAPE
twice
unit
length
unit
length
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
2-D gels: map number & position of replication origins
1st dimension separates by size
2nd
dimension
also
separates
by shape.
twice
unit
length
Simple Y
Fragment
size
doubles
during
replication.
"Bubble-arc"
"Y-arc"
Rela ted to dis tance
from ori to end of
fragme nt.
unit
length
Bubble
Double Y
Asymmetric
Example of analysis of a replicon using 2-D gels
Restriction fragments:
1st dimension separates by size
1st dimension
separates
by size
1st dimension
separates
by size
2nd
dimension
also
separates
by shape.
"Y-arc"
twice
unit
length
"Bubble-arc"
2nd 2nd
"Y-arc"
"Y-arc"
dimension
dimension
also also
separates
separates
by shape.
by shape.
unit
length
twicetwice
unit unit
length
length
1st dimension
separates by size
1st dimension separates
by size
dis
"Bubble-arc"
2ndtance
2nd Rela ted to"Y-arc"
"Bubble-arc"
from ori to dimension
end of
dimension
fragme nt.
also
separates
by shape.
unit unit
length
length
Rela
ted
toted
disto
tance
Rela
dis"Bubble-arc"
tance
"Y-arc"
"Bubble-arc"
from from
ori toori
end
toof
end of
fragme
nt. nt.
fragme
also
separates
by shape.
twice
unit
length
twice
unit
unit
length
length
unit
length
origin
Fork movement
1st dimension separates by size
Rela ted to dis tance
Rela ted
to dis tance
"Bubble-arc"
2nd
"Y-arc"
from ori to end offrom ori to end of
dimension
fragme nt.
fragme
nt.
also
separates
by shape.
twice
unit
length
termination
unit
length
Positions of oriC and ter in E. coli
Forks meet and terminate in this approx. 100 kb region
terD and terA
block progress of
Fork 1
and
terC and terB
block progress of
Fork 2
are 23 bp binding
sites for T us, a
"contra-helicase."
E. coli chromosome
Replication fork 1
Replication fork 2
oriC
245 bp
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
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
61
121
181
241
301
361
GGATCCGGAT
CGGGCCGTGG
AAAAGAAGAT
GCCCTGTGGA
GTGAATGATC
CTCAAAAACT
AGAGTTATCC
AAAACATGGT
ATTCTACTCA
CTATTTATTT
TAACAAGGAT
GGTGATCCTG
GAACAACAGT
ACAGTAGATC
GATTGCCTCG
ACTTTGTCGG
AGAGATCTGT
CCGGCTTTTA
GACCGTATAA
TGTTCTTTGG
GCACGATCTG
CATAACGCGG
CTTGAGAAAG
TCTATTGTGA
AGATCAACAA
GCTGGGATCA
ATAACTACCG
TATACTTATT
TATGAAAATG
ACCTGGGATC
TCTCTTATTA
CCTGGAAAGG
GAATGAGGGG
GTTGATCCAA
TGAGTAAATT
GATTGAAGCC
CTGGGTATTA
GGATCGCACT
ATCATTAACT
TTATACACAA
GCTTCCTGAC
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
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
Proteins needed for initiation at oriC #3
• DnaG primase
• Gyrase
• SSB
• All but DnaA are also used in
elongation
Initiation at oriC: Model
Positions of ter sequences in E. coli
Forks meet and terminate in this approx. 100 kb region
terD and terA
block progress of
Fork 1
and
terC and terB
block progress of
Fork 2
are 23 bp binding
sites for T us, a
"contra-helicase."
E. coli chromosome
Replication fork 1
Replication fork 2
oriC
245 bp
Termination of replication: DNA sites
and proteins needed
• DNA sites: ter sequences, 23 bp
– terD and terA block progress of counterclockwise 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 Sadenosylmethionine 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
m
GATC
CTAG
m
m
GATC
m
GATC
CTAG
CTAG
m
replicate
GATC
CTAG
m
methylate
(lags
behind
replication)
dam methylase
m
GATC
CTAG
m
Fully methylated
Hemimethylated
Fully methylated
Will replicate
Will not replicate
Will replicate