DNA polymerase ␤ (pol ␤) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol ␤ mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol ␤ to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol ␤ mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol ␤ to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol ␤-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol ␤-14 and another of our mutant proteins, pol ␤-166, is probably critical for accurate DNA synthesis by pol ␤. Thus, our genetic method of screening for pol ␤ mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol ␤ enzyme.

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