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DNA polymerase
- looks like a right hand
- palm made of beta sheets and is primary catalytic site
- fingers bind incoming nucleotides and trap the correct dNTP
- thumb stabilizes reaction and processivity by maintaining interaction with the most recent dNTP
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what are the 2 ions used by polymerase to stabilize
- MG++ and ZN++
- first removes a hydrogen and creates a nucleophilic attack on alpha
- second maintains the beta and gamma phosphates
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Operation of exonucleases
- error occurs
- synthesis stops or slows
- exonuclease removes mismatch
- resume synthesis
- only 1 error in 10 to the 10 bps
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addidtion of RNA primers
- RNase makes nick in primer
- exonuclease removes primer
- primer template junction exposed
- polymerase fills junction
- nick joined by ligase
- (primers must be removed from nascent DNA)
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function of Topoisomerase 2
- polymerase reaches super coil
- topoisomerase2 binds and creates either 1 or 2 nicks in DNA
- DNA uncoils and reanneals allowing synthesis to continue
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polymerase alpha:
- same as primase in prokaryotes:
- adds RNA primers
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Polymerase S or E
- they slide with the DNA clamp to extend DNA clamping down the DNA
- pol added at start site of primer and synthesizes DNA untill reaching next primer and falling off
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Start of Replication
- addition of ATP to the clamp loader to activate it by opening it
- bind the sliding clamp to the loader
- begins synthesis of DNA
- hydrolize the ATP on the loader to dessociate the loader
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DNA replication at the fork
- consists of the polymerase core, the T protein with flex linkers, the gamma complex (clamp loader), and sliding clamp
- leadin strand is proccessive and is synthesized continuously from one side to the other
- lagging strand rna primers are added, okazaki fragments are completed, dna pol is released, clamp is loaded on to newly primed lagging strand to synthesize more lagging strands, helicase accosiates with polymerase
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ecoli initaition of replication (prokaryote)
- dnaA and atp mind to origin
- many dnaAs denature dna
- helicase (dnaB) bound by helicase loader (dnaC)
- helicase denatures dna
- primase bound and adds primers
- polymerase added (holoenzyme)
- slidding clamp loaded
- dna synthesized
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eukaryotic replication
- ORC binds to replication origin
- Cdc6 and Cdt1 bind
- Mcm2-7 complex added
- pol B and E replace Cdc6 and Cdt1
- pol A added thus creating primase
- sliding clamp and clamp loader added (PCNA+RF-C)
- initiation of lagging strand synthesis
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CDK activity
- high- existing pre-RC activation/new formation inhibitted
- low- pre-RC formation allowed/ no preRC activation
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mitosis phases
- G1- CDK levels Low
- S- CDK levels high
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resolving dna end
- 3' hang
- telomerase adds primer to 3'
- synthesizes 5'
- repeates many times to extend dna
- synthesizes rest of 3'
- ends once inhibitor proteins cover dna en
- small over hang still persists
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