A GENETIC SYSTEM TO IDENTIFY DNA-POLYMERASE-BETA MUTATOR MUTANTS

DNA polymerase beta (pol beta) is a 39-kDa protein that functions in D NA repair processes in mammalian cells. As a first step toward underst anding mechanisms of polymerase fidelity, we developed a genetic metho d to identify mammalian pol beta mutator mutants. This screen takes ad vantage of a microbial genetics assay and the ability of rat pol beta to substitute for Escherichia coil DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol beta mutat or mutants. Three of the candidate mutator mutants were further charac terized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol beta to our bacterial strain. Pur ification and subsequent analysis of one of our putative mutator prote ins, the pol beta-14 protein, showed that it possesses intrinsic mutat or activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol beta-14 and another of our mutant proteins, pol beta-166, is probably critical fo r accurate DNA synthesis by pol beta. Thus, our genetic method of scre ening for pol beta mutator mutants is useful in identifying active mam malian DNA polymerase mutants that encode enzymes that catalyze DNA sy nthesis with altered fidelity compared with the wild-type pol beta enz yme.