Control of DNA replication Replicon Origins and terminators Solutions to the “end problem”...

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