plan 23. 2. 2004 eukaryot dna replikasjon 1.replikasjonsorigins 2.enzymologi 3.initiering og...
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Plan 23. 2. 2004
Eukaryot DNA replikasjon
1. Replikasjonsorigins
2. Enzymologi
3. Initiering og Regulering av DNA replikasjon
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Chromosomesare denselypacked inmitosis
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The accuracy of DNA replication is seen in the quality of the product
The accuracy of DNA replication is seen in the quality of the product
Duplication of DNA
Cell Division
Fertilised EggProduct
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Components of a
Replication Origin
Components of a
Replication Origin
Replicator
Initiator
PhysicalOrigin
Initiation
“Replicon” = stretch of DNA replicated by the forks from a single origin
(Jacob et al., 1963)
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Autonomously Replicating SequencesAutonomously Replicating Sequences
yeast chromosomal DNA insert
selectable marker gene
LEU2LEU2 LEU2
LEU2
LEU
2
library of different inserts
transfect into leu- yeast
Only yeast containing plasmids with certain sequences will be able to proliferate and form colonies on plates lacking leucine.
This defines “Autonomously Replicating Sequences” or ARSs.
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ACSB1B3 B2
• Budding yeast replication origins map within such ARS elements on both chromosomal and plasmid DNA.
• ARS elements comprise a short 11 bp A element or ‘ARS consensus sequence’: 5’-(A/T)TTTA(T/C)(A/G)TTT(A/T)-3’, plus flanking regions of 100 - 200 bp (‘B’ elements) that enhance origin function.
Characteristics of ARSsCharacteristics of ARSs
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Which proteins bind to and define eukaryotic replication origins?
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ORIGIN RECOGNITION COMPLEX
- ORC ble identifisert som et proteinkompleks sombandt seg til ARS konsensus sekvens.
- ORC består av seks forskjellige proteiner.
- ORC er nødvendig for initiering av replikasjon og er bundet til ARS gjennom hele cellesyklus.
- ORC homologer finnes i alle eukaryoter, til og med i archae
ORC
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Replication origins in metazoans (somatic cells)Replication origins in metazoans (somatic cells)
• The structure of replication origins in higher eukaryotes is unclear.
• Small extrachromosomal DNA sequences replicate poorly, even when carrying >10 kb genomic DNA known to act as origins when in thechromosome.
• Replication initiates at specific regions at a characteristic time in S phase. Both place and timing may change with cell type.
• Replication forks can potentially initiate at a number of different sites throughout an “initiation zone” that may extend over >10 kb.
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The ‘Origin Number’ ParadoxThe ‘Origin Number’ ParadoxE. coli:
Genome, 4 Mb = 4 x 106 bpFork rate approx. 800 bp / secReplication time approx. 40 minutes = 2,400 secs
Amount replicated by 2 forks in 40 mins = 2 x 2400 x 800 = 3,840,000 bp (~4 Mb)
Eukaryotes
Genome 20 Mb (yeast) up to 6,000 Mb (human)Fork rate 10 bp / sec (frog) - 50 bp / sec (mammal)
Amount replicated by 2 forks in 8 hr (human cells) = 2 x 50 x 28,800 = 2,880,000 (~ 3 Mb, a 2,000-fold deficit)
46 chromosomes (human cells) - with one origin per chromosome, at least 92 replication forks gives approx. 140 Mb replicated in 8 hours (still a 40-fold deficit)
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The solution:- eukaryotes replicate their chromosomes from multiple replication origins
Electron micrograph showing an approx. 300 kb stretch of replicating chromosomal DNA from the yeast S. cerevisiae. Replication forks are indicated by an arrow. (Petes, Newlon, Byers, & Fangman 1974;Cold Spring Harb Symp Quant Biol. 38:9-16 ).
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The study of replication origins using DNA fibre autoradiography
The study of replication origins using DNA fibre autoradiography
Interpretation:
Before pulse I:
End of pulse I:
End of pulse II:
Protocol:a) Pulse label proliferating cells with 3H-thymidine for 5 min (pulse I)b) Dilute label to 1/5 activity for further 5 min (pulse II)c) Isolate DNA and spread on a photographic plated) expose for 6 monthse) develop and examine grains under microscope
heavy labellinglight labelling
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Chromosome regions replicate at different timesChromosome regions replicate at different times
BrdU
BrdU
BrdU
BrdU
BrdU
BrdUBrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdUBrdUBrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
BrdU
Protocol:
a) Pulse cells, at different times, with BrdU for 1 hr.
b) “Chase”, collect chromosomes.
c) Stain with anti-BrdU antibodies.
late S2 hr chase
mid S5 hr chase
early S9 hr chase
~ 8 ~2 ~1Duration (hours)
5 hr
9 hr
2 hrG1 S G2 M
= BrdU pulse
= chase
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Organization of replication during S phaseOrganization of replication during S phase
G1
S
G2
template DNA
Typical somatic cell
early-firing origins
late-firing origins
duplicated DNA
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The global pattern of origin usage can also change: eg early embryonic versus somatic cells:
Drosophila somatic cell (transcriptionally active)S phase = 10 hours (600 mins); mean origin spacing = >40kb
Early Drosophila embryo (transcriptionally quiescent)S phase = 3.4 mins; mean origin spacing = 7.9kb
G1
S
G2
near-synchronous initiation
Early Drosophila embryo
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What determines origin usage?
The Jesuit principle:
”Many are called – few are chosen”
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Why so many origins?Why so many origins?
Excess origins are used to lower the probability of a lethal ‘double stall’?
To prevent problems if origins do not initiate with 100% probability?
stalled fork
replication completed byother fork of pair
double stall: no way of replicating intervening DNA
To allow different sections of the genome to replicate at different times?
To allow sections of the genome to replicate faster?
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Facts IFacts I
• Rate of progression of replication forks is fairly constant for a given organism
• Forks generally stop only when they encounter an oppositely moving fork
• Chromosome replication is regulated mainly through control of the initiation of new replication forks
For example:-
-by regulating the number and spacing of origins that fire eg. during development
-by regulating the time during S phase at which different origins are activated
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Facts IIFacts II
• In somatic mammalian cells, most inter-origin distances (replicon sizes) are between 30 - 300 kb (ie would take 5 - 50 min to replicate completely).
• Some adjacent origins (“origin clusters”, typically 2 - 5 origins) initiate synchronously
• Different origins / origin clusters initiate at different times during S phase
Typical mammalian cell replicates 6,000 Mb in 8 hr = 6 x 109 ÷ 28,800 bp/sec ie. ~200,000 bp/sec
For fork rate of 50 bp / sec = 200,000 ÷ 50 ~ 4,000 forks active at any given time in S phase
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Restoration of chromatin after replication
The principle chromatin assembly reactions during DNA replication. Reaction (a): parental nucleosomes are partially disrupted during DNA replication and the histones are directly transferred to the replicated DNA, reassembling into nucleosomes. Reaction (b): the assembly of new nucleosomes from newly synthesized and soluble histones is mediated by a chromatin assembly factor
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PCNA – likhet med ß-subenheten i E.coli pol III
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A eukaryotic DNA replication fork
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Replikasjon av kromosom-ender med telomerase
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Struktur av telomerer: G-kvartett
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Initiering av DNA replikasjon
Regulering av DNA replikasjon
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Initiation of SV40 replication
Initiation of SV40 replicationSV40 T antigen binds and distorts the viral origin.
RP-A (‘replication protein A’) binds to the single-stranded DNA.
DNA polymerase -primase puts down an RNA primer and extends it with DNA.
RF-C displaces pol -primase and loads PCNA to establish the leading strand.
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Trykkfeil: Cdt1, ikke Ctd1
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Cellesyklus for mammalske celler
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Inngang til mitose(Blått: Kromosomer. Grønt: spindel)
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Kromosomene kondenseres
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Spindeltrådene (mikrotubuli) fester seg påkromsomene (sentromerer)
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Kromosomene samles langs metafase-platen
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Mikrotubuli separerer kromosomene
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LICENSING OF DNA REPLICATION
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Somatic cell fusion (Rao and Johnson 1970)Somatic cell fusion (Rao and Johnson 1970)Fuse two cells at different stages of the cell cycle, and track what happens to each of the two nuclei in the first cell cycle following fusion.
Initial FusionProduct
Result Prior to First Mitosis Starting cells
G1+S
G1+
G2
S+
G2
G1 nucleus replicatesearlier than normal
S nucleus finishesreplication normally
G1 nucleus replicates earlier than normal
G2 nucleus does not replicate
S nucleus finishes replication normally
G2 nucleus does not replicate
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Nuclear envelope permeabilisation allows nuclei to re-replicate in Xenopus egg extract
Nuclear envelope permeabilisation allows nuclei to re-replicate in Xenopus egg extract
intact permeable
G1 S G2 M
Isolate and transfer to fresh
extract
re-replication: – ++– –
Blow, J.J. and Laskey, R.A. (1988). Nature 332, 546-548.
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Licensing Factor ModelLicensing Factor Model
( )
( )
( )
( ) MIT
OS
IS
Licensing Factor:
1. Binds tightly to origins
2. Is essential for initiation
3. Is displaced from origins on
initiation/replication
4. Cannot enter an intact nucleus in
active form
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Licensing of
replicationorigins on Xenopus
sperm nuclei
Licensing of
replicationorigins on Xenopus
sperm nuclei
Nucleotide requirement
ADP or ATPORC
N
ORC
Cdt1Cdc6ATP or ATP--S
ORCCdt1Cdc6
M
M MATP hydrolysispre-Replicative
Complex (pre-RC)
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Replication
HsMcm4
Merge
G1 earlyS
midS
late S
Cell CycleStage
Mcm4 in HeLa nucleiMcm4 in HeLa nuclei
Krude et al. (1996). J Cell Sci 109, 309-318.
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Mcm2-7Mcm2-7
Mcm2-7 (mini-chromosome maintenance) proteins were originally identified in yeast because as mutants affecting replication origin usage.
Fractionation showed them to be a key component of Licensing Factor.
They are loaded onto DNA in anaphase and are removed from chromatin during S phase.
They form a hexameric ring, capable of encircling double-stranded DNA.
Highly conserved throughout eukaryotes; archaea also possess an Mcm2-7 homologue
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Mcm proteins have weak helicase activityMcm proteins have weak helicase activity
OH*P
5' 3'
Ishimi Y. (1998). J. Biol. Chem. 272, 24508-13
Mcm(4,6,7)
24-mer
37-merheat-
denatured24-mer
37-mer
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Do Mcm2-7 provide the fork helicase?Do Mcm2-7 provide the fork helicase?
1. Mcm proteins have weak helicase activity (can unwind double-stranded DNA.
2. DNA synthesis stops rapidly if Mcm2-7 proteins are degraded.
3. Chromatin immunoprecipitation shows Mcm2-7 proteins at the fork.
But....
4. There is ~20-fold excess of Mcm2-7 over origins.
5. Immunofluorescence shows no major co-localisation of Mcm2-7 and sites of DNA synthesis.
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Somatic nuclei + egg cytoplasmSomatic nuclei + egg cytoplasm
M G1 S
Isolate nuclei, transfer to Xenopus egg extracts and examine replication.
Gilbert, D.M. et al (1995). Mol. Cell. Biol. 15, 2942-2954.Wu, J.R. and D.M. Gilbert (1996). Science 271, 1270-1272.
Synchronised CHO tissue culture cells
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CHO nuclei become licensed for replication within 1 hr of metaphase exit
CHO nuclei become licensed for replication within 1 hr of metaphase exit
Dimitrova, D.S. et al (2002). J. Cell Sci. 115, 51-59.
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”START” in budding yeast
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The restriction point is when entry into S phase becomes independent of further growth factor stimulation, probably representing activation of the E2F transcription system.
Examples of E2F-regulated genes:
- cyclins A, E and D - CDK1 and CDK2- CDC6 - thymidine kinase- dihydrofolate reductase - DNA polymerase
The Restriction point and the Retinoblastoma protein
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The restriction point
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M G1 S G2 Mmeta- anaphase
Activities required to control chromosome duplicationActivities required to control chromosome duplication
OriginRecognition
(ORC)
Initiation(Cdks + Cdc7)
Licensing(Mcm2-7loading)
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Early S -euchromatin
Replication profile of a typical somatic cell:
Mid S -peripheral heterochromatin
Late S - nucleolar DNA
~ 8 hr
Early S -euchromatin
Replication profile of a typical somatic cell:
Mid S -peripheral heterochromatin
Late S - nucleolar DNA
~ 8 hr
In somatic cells, transcriptionally active euchromatin replicates early, transcriptionally inactive whilst heterochromatin replicates late.
No heterochromatic regions are typically seen in the nuclei of the early Xenopus embryo.
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2D-elektroforese for kartlegging av replikasjonsorigi. Nøytral gel
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Reaksjoner som katalyseres av revers transkriptase
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