TY - JOUR
T1 - A genetic system to identify DNA polymerase β mutator mutants
AU - Washington, Stacy L.
AU - Yoon, Margaret S.
AU - Chagovetz, Alexander M.
AU - Li, Shu Xia
AU - Clairmont, Caroline A.
AU - Preston, Bradley D.
AU - Eckert, Kristin A.
AU - Sweasy, Joann B.
PY - 1997/2/18
Y1 - 1997/2/18
N2 - 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.
AB - 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.
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U2 - 10.1073/pnas.94.4.1321
DO - 10.1073/pnas.94.4.1321
M3 - Article
C2 - 9037051
AN - SCOPUS:0031029917
SN - 0027-8424
VL - 94
SP - 1321
EP - 1326
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 4
ER -