DNA Replication

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

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?

• 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


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


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)


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


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


Origin Redundancy vs fixed spacing

• Many more potential than actual origins

• Passive inactivation via replication

• Possible lateral inhibition of activation

• Mechanism?


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


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

DNA Replication

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