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