Week9-1 DNA Replication I

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Eukaryotic chromosome maintenance

Monocentric chromosomes – common theme Holocentric chromosome (C. elegans) – rare case

DNA REPLICATION

Chapter 8 +

Additional materials

Matthew Meselson and Franklin Stahl

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Semi-conservative mode of DNA replication (1958)

Matthew Meselson and Franklin Stahl

“The most beautiful experiment in biology”

(Chapter 2)

Tout ce qui est vrai pour le Colibacille est vrai pour l’elephant.

[ All that true for E.coli is true for the elephant.]

Jacques Monod

Tout ce qui est vrai pour le levure de bière est vrai pour l’elephant.

[ All that true for yeast is true for the elephant.]

165 genes are responsible for replication of the human genome (only 3 are absent in yeast)

40 diseases are caused by mutations in genes required for DNA

replication

14 therapeutic drugs

Aging!

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The replicon model (Jacob et al. 1963,1964)

DEFENITIONS: Replicator –a genetic element required for initiation of DNA replication Replicon – the unit of DNA whose duplication is controlled through replicator Initiator – a protein or protein complexes that bind replicator and are

required for initiation Initiation site – a replication origin

The replicon model (Jacob et al. 1963,1964)

DEFENITIONS: Replicator –a genetic element required for initiation of DNA replication Replicon – the unit of DNA whose duplication is controlled through

replicator Initiator – a protein or protein complexes that bind replicator and are

required for initiation Initiation site – a replication origin. A location on chromatin, from which

replication fork emanate

Function of the initiator proteins during initiation of DNA replication

Initiator – origin recognition complex (ORC), 6 proteins (requires ATP hydrolysis)

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Eukaryotic cells have multiple origins of replication

A replicon is a unit of replication

Organism Replicons Length

E.coli 1 4639 kb

Yeast 500 40 kb

Fruit fly 3,500 40 kb

Toad 15,000 50-200 kb

Mouse 25,000 50-150 kb

Human 30,000 50-150 kb

Structure of replicators in yeast

Green – sites that bind origin recognition complex (ORC) Blue – sites initiating unwinding (AT-rich) + binding site for MCM2-7 helicase Red – start of DNA synthesis

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Structure of replicators in yeast

Green – sites that bind origin recognition complex (ORC-6 subunits) Blue – sites initiating unwinding (AT-rich) Red – start of DNA synthesis

Autonomosly replicating sequences (ARS elements) =

Saccharomyces cerevisiae

Schizosaccharomyces pombe

Homo sapiens

Replication origins in eukaryotes

130 bp

ARS305

ars1

800 bp

initiation

c-myc

2.4 kb

AT-rich region

AT-rich blocks

6 subunits of ORC

ORC4 9 AT-hook motifs Most of the replicator sites are located in intergenic regions

(have open, nucleosome free chromatin)

also often include transription-factor-binding sites

such as Abf1 and Fkh1-Fkh2

A1 B1

A1 – (A/T)TTTAYRTTT(A/T)

EM structures of the yeast ORC complex

Chen Z et al. PNAS 2008;105:10326-10331 ©2008 by National Academy of Sciences

Winged helix DNA binding domans (WHD) from 5 Orcs specifically interact with A and B1 sequence regions

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Homo sapiens


130 bp

ARS305

ars1

800 bp

initiation

c-myc

2.4 kb

AT-rich region

AT-rich blocks

6 subunits of ORC

ORC4 9 AT-hook motifs



Homo sapiens


130 bp

ARS305

ars1

800 bp

initiation

c-myc

2.4 kb

AT-rich region

AT-rich blocks

6 subunits of ORC

ORC4 9 AT-hook motifs

Only 40 human origins have been identified so far

EUKARYOTIC CHROMOSOMES HAVE MULTIPLE REPLICATORS

A REPLICATOR SHOULD BE ACTIVATED ONLY ONCE THROUGH S-PHASE NOT ALL REPLICATORS ARE ACTIVATED IN EVERY ROUND OF EUKARYOTIC DNA REPLICATION

Replication of replicator inactivates it

ARS elements do not always function as origins

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Distribution of ARS elements on chromosome III

Different origins have different efficiency of firing


ARS Origin !

There are 1200 ARS consensus sequences in the yeast genome but only ~400 are used as origins


late

early

Different origins have different timing of firing

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Replication timing model based on spatial organization of origins and limiting factor accumulation

Aparicio O M Genes Dev. 2013;27:117-128 ©2013 by Cold Spring Harbor Laboratory Press

Fkh1/2-bound origins create clusters that localize with Cdc45

Chromosome breakage as a result of incomplete DNA replication

Yeast cell cycle

G1 S G2

M

S

G1 G2

Replication

Segregation

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REPLICATOR SELECTION OCCURS IN G1 PHASE BY ASSEMBLY OF PRE-REPLICATIVE COMPLEX (PRE - RC)

Cdc6 and Cdt1 – helicase-loading proteins Mcm2-7 – helicase complex

PRE-RC ACTIVATION OCCURS IN THE S-PHASE VIA PHOSPHORYLATION OF SOME PRE-RC COMPONENTS BY PROTEIN KINASES Cdk AND Ddk

PROTEIN KINASES ARE PROTEINS THAT PHOPHORYLATE OTHER PROTEINS

Cdk and Ddk are not active before S-phase

PHOSPHORYLATION CHANGES AFFINITY OF SOME PRE-RC COMPONENTS such as Cdc45p and GINS ONCE COMPOSITION OF PRE-RC IS CHANGED, IT CAN BIND AUXILLIARY FACTORS AND POLYMERASES, AND BEGIN DNA UNWINDING AND SYNTHESIS

Cdc45 GINS

G1

S, G2 and M phase

Cyclin dependent kinases (Cdk) are required to activate pre-RCs and to inhibit the formation of new pre-RCs

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The minimal set of proteins for eukaryotic replication fork

The updated set of proteins at the replication fork

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REPLICATOR SELECTION OCCURS IN G1 PHASE BY ASSEMBLY OF PRE-REPLICATIVE COMPLEX (PRE - RC)

Cdc6 and Cdt1 – helicase-loading proteins Mcm2-7 – helicase complex

DNA helicase separates the two strands of the DNA duplex

Mcm2-7 complex

Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7

Mcm10 coordinates interaction between the helicase and the Polα

Mcm2-7 is 3’-5’ helicase

Mcm2-7 complex

Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7

Mcm10 coordinates interaction between the helicase and the Polα



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A biochemical assay for DNA helicase activity

Bochman ML, Schwacha A. Mol Cell. 2008 Jul 25;31(2):287-93.

Mcm2-7 is loaded onto DNA as a head-to-head double hexamer

Remus et al., 2009, Cell

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Biochem. J. (2010) 425, 489-500

GINS ( go-ichi-ni-san ~ Sld5, Psf1,Psf2 and Psf3) complex

Preferentially binds ss-DNA

GINS and Cdc45 are required to jump –start MCM2-7 helicase

Helicase loading and action

Fu et al., Cell. 2011

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Cooperative binding of single-stranded binding protein RP-A to DNA

RP-A complex Rpa14 Rpa32 Rpa70

binds ~ 28-30 nt


Topoisomerases remove supercoils produced by DNA unwinding at the replication fork

topoisomerase I and

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palm

Structural organization of DNA polymerases

Documents

Week9-1 DNA Replication I