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