4.5.rep.forks - DNA polymerase summary 1....

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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  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 III 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.
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4.5.rep.forks - DNA polymerase summary 1....

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