A genetic system to identify DNA polymerase β mutator mutants

Stacy L. Washington, Margaret S. Yoon, Alexander M. Chagovetz, Shu Xia Li, Caroline A. Clairmont, Bradley D. Preston, Kristin A. Eckert, Joann B. Sweasy

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Abstract

DNA polymerase β (pol β) 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 β mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol β to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol β 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 β to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol β-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 β-14 and another of our mutant proteins, pol β-166, is probably critical for accurate DNA synthesis by pol β. Thus, our genetic method of screening for pol β 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 β enzyme.

Original languageEnglish (US)
Pages (from-to)1321-1326
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number4
DOIs
Publication statusPublished - Feb 18 1997

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All Science Journal Classification (ASJC) codes

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Washington, S. L., Yoon, M. S., Chagovetz, A. M., Li, S. X., Clairmont, C. A., Preston, B. D., ... Sweasy, J. B. (1997). A genetic system to identify DNA polymerase β mutator mutants. Proceedings of the National Academy of Sciences of the United States of America, 94(4), 1321-1326. https://doi.org/10.1073/pnas.94.4.1321