A genetic system to identify DNA polymerase beta mutator mutants.
DNA polymerase beta (pol beta) 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 beta mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol beta to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol beta 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 beta to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol beta-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 beta-14 and another of our mutant proteins, pol beta-166, is probably critical for accurate DNA synthesis by pol beta. Thus, our genetic method of screening for pol beta 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 beta enzyme.
Washington
S L
SL
Department of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.
Yoon
M S
MS
Chagovetz
A M
AM
Li
S X
SX
Clairmont
C A
CA
Preston
B D
BD
Eckert
K A
KA
Sweasy
J B
JB
D000818:Animals; D004256:DNA Polymerase I / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D016296:Mutagenesis / Q000235:genetics*; D010641:Phenotype; D051381:Rats; D011994:Recombinant Proteins; D012641:Selection, Genetic; D014443:Tyrosine / Q000235:genetics
D011994:Recombinant Proteins; D014443:Tyrosine; D004256:DNA Polymerase I