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Plan 23. 2. 2004 Eukaryot DNA replikasjon Replikasjonsorigins Enzymologi Initiering og Regulering av DNA replikasj

Plan 23. 2. 2004 Eukaryot DNA replikasjon Replikasjonsorigins Enzymologi Initiering og Regulering av DNA replikasjon. Chromosomes are densely packed in mitosis. Fertilised Egg. Product. The accuracy of DNA replication is seen in the quality of the product. Replicator.

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Plan 23. 2. 2004 Eukaryot DNA replikasjon Replikasjonsorigins Enzymologi Initiering og Regulering av DNA replikasj

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  1. Plan 23. 2. 2004 • Eukaryot DNA replikasjon • Replikasjonsorigins • Enzymologi • Initiering og Regulering • av DNA replikasjon

  2. Chromosomes are densely packed in mitosis

  3. Fertilised Egg Product The accuracy of DNA replication is seen in the quality of the product

  4. Replicator Components of a Replication Origin Initiator “Replicon” = stretch of DNA replicated by the forks from a single origin Physical Origin (Jacob et al., 1963) Initiation

  5. yeast chromosomal DNA insert library of different inserts transfect into leu- yeast selectable marker gene Autonomously Replicating Sequences 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.

  6. • 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. ACS B1 B3 B2 Characteristics of ARSs

  7. Which proteins bind to and define eukaryotic replication origins?

  8. ORC • ORIGIN RECOGNITION COMPLEX • ORC ble identifisert som et proteinkompleks som • bandt 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

  9. 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 the chromosome. • 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.

  10. The ‘Origin Number’ Paradox E. coli: Genome, 4 Mb = 4 x 106 bp Fork rate approx. 800 bp / sec Replication 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)

  11. 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 ).

  12. heavy labelling light labelling Interpretation: Before pulse I: End of pulse I: End of pulse II: The study of replication origins using DNA fibre autoradiography 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 plate d) expose for 6 months e) develop and examine grains under microscope

  13. Duration (hours) ~ 8 ~2 ~1 G1 S G2 M 2 hr 5 hr 9 hr = chase = BrdU pulse Chromosome regions replicate at different times • Protocol: a) Pulse cells, at different times, with BrdU for 1 hr. • b) “Chase”, collect chromosomes. • c) Stain with anti-BrdU antibodies. BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU BrdU late S 2 hr chase mid S 5 hr chase early S 9 hr chase

  14. Typical somatic cell template DNA early-firing origins late-firing origins duplicated DNA Organization of replication during S phase

  15. Early Drosophila embryo near-synchronous initiation 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

  16. What determines origin usage? The Jesuit principle: ”Many are called – few are chosen”

  17. stalled fork replication completed by other fork of pair double stall: no way of replicating intervening DNA Why so many origins? To allow sections of the genome to replicate faster? To allow different sections of the genome to replicate at different times? To prevent problems if origins do not initiate with 100% probability? Excess origins are used to lower the probability of a lethal ‘double stall’?

  18. 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

  19. 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

  20. 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

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

  22. A eukaryotic DNA replication fork

  23. Replikasjon av kromosom-ender med telomerase

  24. Struktur av telomerer: G-kvartett

  25. Initiering av DNA replikasjon Regulering av DNA replikasjon

  26. Initiation of SV40 replication SV40 T antigen binds and distorts the viral origin. RP-A (‘replication protein A’) binds to the single-stranded DNA. DNA polymerase a -primase puts down an RNA primer and extends it with DNA. RF-C displaces pol a-primase and loads PCNA to establish the leading strand.

  27. Trykkfeil: Cdt1, ikke Ctd1

  28. Cellesyklus for mammalske celler

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

  30. Kromosomene kondenseres

  31. Spindeltrådene (mikrotubuli) fester seg på kromsomene (sentromerer)

  32. Kromosomene samles langs metafase-platen

  33. Mikrotubuli separerer kromosomene

  34. LICENSING OF DNA REPLICATION

  35. 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 Fusion Product Result Prior to First Mitosis Starting cells G1 nucleus replicates earlier than normal S nucleus finishes replication normally G1 + S G1 + G2 G1 nucleus replicates earlier than normal G2 nucleus does not replicate S nucleus finishes replication normally G2 nucleus does not replicate S + G2

  36. G1 S G2 M Isolate and transfer to fresh extract intact permeable – – – + + re-replication: Nuclear envelope permeabilisation allows nuclei to re-replicate in Xenopus egg extract Blow, J.J. and Laskey, R.A. (1988). Nature 332, 546-548.

  37. MITOSIS Licensing Factor Model 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

  38. Nucleotide requirement N ADP or ATP ORC Cdt1 Cdc6 ATP or ATP-g-S ORC M ATP hydrolysis Cdt1 Cdc6 ORC M M pre-Replicative Complex (pre-RC) Licensing ofreplicationorigins on Xenopussperm nuclei

  39. Replication HsMcm4 Merge early S mid S late S Cell Cycle Stage G1 Mcm4 in HeLa nuclei Krude et al. (1996). J Cell Sci 109, 309-318.

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