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ibs602_rodgers_2_opt

ibs602_rodgers_2_opt - replication/elongation active site...

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replication/elongation active site structure is conserved among polymerases central sheet, flanking helices, catalytic aspartate residues E. coli pol I T7 RNA pol HIV-1 RT pol !
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replication/elongation nucleic acid binding by polymerases HIV reverse transcriptase Arnold et al. (1992) Nature 357, 85.
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replication/elongation two metal ion mechanism of polymerase catalysis
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replication/elongation visualizing the catalytic mechanism Double et al. (1998) Nature 391, 251.
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replication/elongation substrate specificity Fig. 8-3a Watson
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replication/elongation Fig. 8-7a Watson induced fit -- movement of fingers domain
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replication/elongation fidelity of replication error rate in E. coli is about one mispairing in 10 8 to 10 10 pairs corresponds to about 1 error per 1000 bacteria per generation How is such a low error rate achieved? 1. balanced levels of dNTPs 2. high fidelity polymerases accurate placement for tight binding and catalysis requires Watson-Crick pairing 3. proofreading function enzyme pauses and excises incorrect base 4. repair of mismatches and other errors
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replication/elongation editing steps in polymerase catalysis 1. primer/template binding 2. dNTP binding 3. conformational change 4. catalytic step 5. PPi release? 6. translocation? Joyce and Benkovic (2004) Biochemistry 43, 14317.
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replication/elongation E. coli DNA pol I Klenow fragment editing 3’-->5’ exonuclease activity of pol I Beese et al. (1993) Science 260, 352.
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replication/elongation overview Watson Fig. 8-26 replisome
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replication/elongation replication cycle on lagging strand
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