Genetics. Published Articles Ahead of Print: February 3, 2005, Copyright © 2005
doi:10.1534/genetics.104.037630


A more recent version of this article appeared on April 1, 2005.

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Processivity Clamp gp45 and ssDNA-Binding-Protein gp32 Modulate the Fidelity of Bacteriophage RB69 DNA Polymerase in a Sequence-Specific Manner, Sometimes Enhancing and Sometimes Compromising Accuracy

Anna Bebenek 1, Geraldine T. Carver 2, Farid A. Kadyrov 3, Grace E. Kissling 2 and John W. Drake 4*

1 Polish Academy of Science
2 National Institute of Environmental Health Sciences
3 Duke University
4 National Inst. Environmental Health Sciences

* To whom correspondence should be addressed. E-mail: drake{at}niehs.nih.gov.

Submitted on October 15, 2004
Revised on December 1, 2004
Accepted on 21 December 2004


Abstract

Numerous studies of the impacts of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZa mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading, and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased Ttemplate•Gprimer mispairs. Evidence was found for many additional sites where the accessory proteins impact fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.

Key Words: DNA polymerase accessory proteins, DNA polymerase fidelity, bacteriophage RB69




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