of Work: Extensive genetic studies have established that the fidelities of replicative DNA polymerases and their associated proofreading 3'-exonucleases are the primary determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have developed a bacteriophage system that permits the rapid analysis of the fidelities of mutant polymerases in vivo without a requirement for enzyme over-expression, purification, and fidelity analysis in vitro. Bacteriophage RB69 DNA polymerase (whose structure has been described) supplied from a plasmid can replace the normal bacteriophage T4 DNA polymerase when the latter is mutationally inactivated, retaining high fidelity during T4 DNA replication. Using mutations that alter critical polymerase and exonuclease amino acids, we are characterizing the resulting changes in mutation rates using both reversion and forward-mutation tests in vivo and in vitro. Either inactivating the proofreading exonuclease or altering a key tyrosine residue in the polymerase active site strongly increases mutation rates. When the fidelities of the mutant polymerases are examined in vitro with or without the support of accessory proteins (the SSB and the clamp), overall impacts of the accessory proteins on fidelity are nil or small but large impacts are readily observed at numerous specific sites, sometimes increasing and sometimes decreasing mutation rates.
