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LECTURE 4&5 - The DNA replication fork collaborate or die-Other replication factors it takes a v

LECTURE 4&5 - The DNA replication fork collaborate or die-Other replication factors it takes a v

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DNA polymerase summary 1. DNA replication is semi-conservative. 2. DNA polymerase enzymes are specialized for different functions. 3. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease. 4. DNA polymerase structures are conserved. 1. But: Pol can’t start and only synthesizes DNA 5’-->3’! 1. Editing (proofreading) by 3’-->5’ exo reduces errors. 2. High fidelity is due to the race between addition and editing. 3. Mismatches disfavor addition by DNA pol I at 5 successive positions. The error rate is ~ 1/10 9 .
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Replication fork summary 1. DNA polymerase can’t replicate a genome. Problem Solution ATP? No single stranded template Helicase + The ss template is unstable SSB (RPA (euks)) - No primer Primase (+) No 3’-->5’ polymerase Replication fork Too slow and distributive SSB and sliding clamp - 2. Replication fork is organized around an asymmetric, DNA- polymerase II I dimer. 3. Both strands made 5’-->3’. 4. “Leading strand” is continuous; “lagging strand” is discontinuous.
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DNA polymerase can’t replicate a genome! 1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive
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Solution: the replication fork 1. No single-stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive Schematic drawing of a replication fork
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DNA polymerase holoenzyme QuickTimeª and a DV - PAL decompressor are needed to see this picture.
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DNA replication factors were discovered using “temperature sensitive” mutations 1. No single stranded template 1. The ss template is unstable 2. No primer 3. No 3’-->5’ polymerase. 4. Too slow in vitro. Mutations that inactivate the DNA replication machinery are lethal. Temperature sensitive (conditional) mutations allow isolation of mutations in essential genes. 37 º C 42 º C 42 º C, Mutant gene overexpressed
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A hexameric replicative helicase unwinds DNA ahead of the replication fork 1. No single stranded template 1. The ss template is unstable 2. No primer 3. No 3’-->5’ polymerase. 4. Too slow in vitro. Replicative DNA helicase is called DnaB in E. coli .
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