Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins.

Abstract:

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant (Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-). Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes revealed the patterns and efficiencies of proofreading, while Tyr(567) was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.

Polymerases:

Topics:

Mutational Analysis, Fidelity, Accessory Proteins/Complexes, Nucleotide Incorporation, Exonuclease Activity

One line summary:

By observing the mutation rate of three RB69 mutants within in vivo and in vitro environments, it can be determined that accessory proteins play a significant role in either increasing or decreasing mutation fidelity

Status:

new topics/pols set partial results complete validated

Results:

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