Lecture15 - Outline DNA replication -basics if replication...

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458 Outline DNA replication -basics if replication -replication machinery: the replisome -Define terms, scope of the challenge in different organisms -Origins of replication and coupling replication to the cell cycle Telomeres -defining chromosome ends -end-replication problem => telomerase -telomeres and aging
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459 Review DNA replication. .. -is semi-conservative -is always directed 3’ => 5’ (by the template strand ), synthesized 5’ => 3’ -requires a nucleic acid primer with a 3’-OH (DNA or RNA) -is fundamentally different for each strand ( leading vs. lagging ) -is mechanistically similar in prokaryotes and eukaryotes
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460 Starting DNA synthesis: Process: Machinery: recognize the origin form an open complex synthesize a primer extend the primer
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461 Replisome: What is the machinery? DNA polymerase processivity factor helicase primase clamp loader exonuclease ssDNA binding protein RNase H ligase http://www. youtube .com/watch? v=4jtmOZaIvS0 &feature=related
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462 Sliding Clamp processivity factor ± binds ² and ³ ´ binds SSB ² Clamp Loader binds ³ and µ µ ' ³ complex => binds ± µ binds ATP ³ dimerizes core binds ³ complex stimulates · ¸ core 3’->5’ exonuclease · DNA polymerase ¹ The E. coli DNA polymerase III holoenzyme contains multiple copies of seven different subunits:
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463 Sliding clamp = Processivity factor -completely encircles DNA -binds DNA polymerase -keeps DNA Pol "locked in" ± how does the clamp get onto the DNA? clamp loader complex
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464 Coordinating lagging strand synthesis: dimeric clamp loader
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465 RNA primer is removed by an RNAseH and gap is filled in and Okazaki fragments are ligated
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466
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467 How to balance efficiency with accuracy? efficiency -helicase -processivity factor => sliding clamp 3 kBp/min 3 kBp/min 30 kBp/min Fork Speed S would last 1 year if only 1 ori >10,00 0 7 hr1 3000 MBp Human S would last 80 hr if only 1 ori ~330 20 min 14 MBp Yeast S longer than doubling time 1 40 min 4.6 MBp E. coli Comments Origins S-phase Genome How long will it take E. coli to replicate DNA? A. ~ 5 hours B. ~2.5 hours C. ~75 minutes D. ~20 minutes
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Accuracy (Fidelity) The error rate of DNA polymerase is ~1/100,000 bp 3 kBp/min 3 kBp/min 30 kBp/min Fork Speed S would last 1 year if only 1 ori >10,00 0 7 hr1 3000 MBp Human S would last 80 hr if only 1 ori ~330 20 min 14 MBp Yeast S longer than doubling time 1 40 min 4.6 MBp E. coli Comments Origins S-phase Genome How many errors for one round of yeast replication? A. ~ 14
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This note was uploaded on 03/28/2010 for the course MCD BIO 110 taught by Professor Hinck during the Fall '09 term at University of California, Santa Cruz.

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Lecture15 - Outline DNA replication -basics if replication...

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