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DNA Replication

DNA Replication. MCM proteins and “random completion”. DNA replicates once and only once. How is this done? Requires multiple origins Requires control of origin density Requires regulated origin activation Requires NO specific DNA sequence. How to ensure ALL DNA replicated?.

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DNA Replication

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  1. DNA Replication MCM proteins and “random completion” Karp/CELL & MOLECULAR BIOLOGY 3E

  2. DNA replicates once and only once • How is this done? • Requires multiple origins • Requires control of origin density • Requires regulated origin activation • Requires NO specific DNA sequence Karp/CELL & MOLECULAR BIOLOGY 3E

  3. How to ensure ALL DNA replicated? • Some cells may delay entry to M: S/M checkpoint • Not necessary in vast majority of replications • Frogs, fish, insects • S then M then S then M ... • No G1, no G2 • No S/M checkpoint • Damage generally ignored • But still: normal development is prevalent Karp/CELL & MOLECULAR BIOLOGY 3E

  4. Orcs and MCM’s • Orcs help load MCM helicase onto DNA • “Licensing” happens late M and G1 • requires CDC6, CDT1 • Geminen and CDK’s stop licensing after S • After mcm’s loaded, cdc6, cdt1, orcs not needed • Activation done by CDC45, CDC7/DBF4, CDK’s Karp/CELL & MOLECULAR BIOLOGY 3E

  5. Karp/CELL & MOLECULAR BIOLOGY 3E

  6. MCM paradox • Mcm’s excluded from replicated chromatin • Most mcm’s localized on unreplicated DNA • Mcm’s and orcs do not colocalize • Mcm’s greatly outnumber orcs (10-100 fold) Karp/CELL & MOLECULAR BIOLOGY 3E

  7. Timing • Origin specification occurs after licensing • Not sequence specific • Not all origins fire at same time • Frequency of firing is stable or increases during S Karp/CELL & MOLECULAR BIOLOGY 3E

  8. Completion problem • In frogs, each bubble can cover only ~20kb! • Average spacing less than 10kb • Closer if random, asynchronous activation • Spacing of ori’s must be more regular than random • Otherwise, a significant probability of > 20kb spacing Karp/CELL & MOLECULAR BIOLOGY 3E

  9. Origin Redundancy vs fixed spacing • Many more potential than actual origins • Passive inactivation via replication • Possible lateral inhibition of activation • Mechanism? Karp/CELL & MOLECULAR BIOLOGY 3E

  10. Karp/CELL & MOLECULAR BIOLOGY 3E

  11. Evidence for Non-random • Eye-length and eye size observed via EM • Excess of origins spaced by 10 kb (vs random) • Recycling of limiting activation component • Excluded from replicated DNA • Hence, targets decrease, activation rate increases Karp/CELL & MOLECULAR BIOLOGY 3E

  12. Karp/CELL & MOLECULAR BIOLOGY 3E

  13. Model • Orcs load multiple mcm’s, each a potential origin • Cdc45 (and others) activate a fraction of mcm’s randomly • Lateral inactivation (? At least partly by replication) provides excess of well spaced ori’s Karp/CELL & MOLECULAR BIOLOGY 3E

  14. Karp/CELL & MOLECULAR BIOLOGY 3E

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