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3/3/14
1
Eukaryotic chromosome maintenance
Monocentric chromosomes – common theme Holocentric chromosome (C. elegans) – rare case
DNA REPLICATION
Chapter 8 +
Additional materials
Matthew Meselson and Franklin Stahl
3/3/14
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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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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
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
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
Distribution of ARS elements on chromosome III
ARS Origin !
There are 1200 ARS consensus sequences in the yeast genome but only ~400 are used as origins
Distribution of ARS elements on chromosome III
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
3/3/14
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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
Mcm2-7 is 3’-5’ helicase
Mcm2-7 is 3’-5’ helicase
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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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The updated set of proteins at the replication fork
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
The updated set of proteins at the replication fork
Topoisomerases remove supercoils produced by DNA unwinding at the replication fork
topoisomerase I and
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palm
Structural organization of DNA polymerases