GDCB 511-4-15-09a

GDCB 511-4-15-09a - GDCB 511: 15 April 2009 DNA Replication...

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GDCB 511: 15 April 2009 DNA Replication in Prokaryotes (continued) Major proteins involved in DNA replication in E . coli Three stages of DNA replication
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DNA Polymerases in E. coli : PolI, PolII, PolIII PolI: DNA polymerase I discovered by Arthur Kornberg in 1958. Holoenzyme: 102 kDa polypeptide, with 3 activities: 5’ – 3’ DNA polymerase 3’ – 5’ exonuclease for proofreading; removes mismatched bases 5’ – 3’ exonuclease degrades DNA (or RNA) ahead of the advancing polymerase. Functions in removal of RNA primers and in DNA repair
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5’ 3’ exonuclease activity of DNA PolI This activity allows pol I to degrade a strand ahead of advancing polymerase Removes and replaces a strand in one pass Basic functions are: Primer removal Nick repair
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Polymerases II and III Pol II activity is not required for DNA replication Pol I appears mostly active in repair Only pol III is required for DNA replication Pol III is the enzyme that replicates bacterial DNA
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The Pol III Holoenzyme Pol III core is composed of 3 subunits: DNA polymerase activity is in the α -subunit 3’ 5’exonuclease activity found in ε -subunit Not yet clear what is the role of θ -subunit DNA-dependent ATPase activity is located in the γ -complex containing 5 subunits Lastly, β -subunit plus the other 8 comprise the holoenzyme
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PolIII holoenzyme contains ten different polypeptides
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Strand Separation DNA replication assumes that the 2 DNA strands at the fork somehow unwind Does not happen automatically as DNA polymerase does its job 2 parental strands hold tightly to each other This takes energy and enzyme action to separate them Helicase that unwinds dsDNA at the replicating fork is encoded by E. coli dnaB gene
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Fig. 20.21 Helicase activity of DnaB
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GDCB 511-4-15-09a - GDCB 511: 15 April 2009 DNA Replication...

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