Plan 23. 2. 2004

Eukaryot DNA replikasjon

1. Replikasjonsorigins

2. Enzymologi

3. Initiering og Regulering av DNA replikasjon

Chromosomesare denselypacked inmitosis

The accuracy of DNA replication is seen in the quality of the product

Duplication of DNA

Cell Division

Fertilised EggProduct

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)

Autonomously Replicating Sequences

yeast chromosomal DNA insert

selectable marker gene

LEU2LEU2 LEU2

LEU2

LEU2

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.

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 ARSs

Which proteins bind to and define eukaryotic replication origins?

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

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.

The ‘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)

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 ).

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

Chromosome regions replicate at different times

BrdU

BrdU

BrdUBrdU

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

Organization of replication during S phase

G1

S

G2

template DNA

Typical somatic cell

early-firing origins

late-firing origins

duplicated DNA

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

What determines origin usage?

The Jesuit principle:

”Many are called – few are chosen”

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?

Facts 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

Facts 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

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

PCNA – likhet med ß-subenheten i E.coli pol III

A eukaryotic DNA replication fork

Replikasjon av kromosom-ender med telomerase

Struktur av telomerer: G-kvartett

Initiering av DNA replikasjon

Regulering av DNA replikasjon

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.

Trykkfeil: Cdt1, ikke Ctd1

Cellesyklus for mammalske celler

Inngang til mitose(Blått: Kromosomer. Grønt: spindel)

Kromosomene kondenseres

Spindeltrådene (mikrotubuli) fester seg påkromsomene (sentromerer)

Kromosomene samles langs metafase-platen

Mikrotubuli separerer kromosomene

LICENSING OF DNA REPLICATION

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

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.

Licensing Factor Model

( )

( )

( )

( ) MIT

OSI

S

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

Licensing of

replicationorigins on Xenopus

sperm nuclei

Nucleotide requirement

ADP or ATPORC

N

ORCCdt1Cdc6

ATP or ATP--S

ORCCdt1Cdc6

M

M MATP hydrolysispre-Replicative

Complex (pre-RC)

Replication

HsMcm4

Merge

G1 earlyS

midS

late S

Cell CycleStage

Mcm4 in HeLa nuclei

Krude et al. (1996). J Cell Sci 109, 309-318.

Mcm2-7

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

Mcm 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

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.

Somatic 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

CHO nuclei become licensed for replication within 1 hr of metaphase exit

Dimitrova, D.S. et al (2002). J. Cell Sci. 115, 51-59.

”START” in budding yeast

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

The restriction point

M G1 S G2 Mmeta- anaphase

Activities required to control chromosome duplication

OriginRecognition

(ORC)

Initiation(Cdks + Cdc7)

Licensing(Mcm2-7loading)

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.

2D-elektroforese for kartlegging av replikasjonsorigi. Nøytral gel

Reaksjoner som katalyseres av revers transkriptase