1. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E DNA Replication MCM proteins and “random completion”

2. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

3. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

4. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

5. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

6. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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)

7. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

8. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

9. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Origin Redundancy vs fixed spacing Many more potential than actual origins Passive inactivation via replication Possible lateral inhibition of activation Mechanism?

10. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

11. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

12. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

13. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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

14. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E