A genetic system to identify DNA polymerase β mutator mutants
| Autor: | Joann B. Sweasy, Caroline A. Clairmont, Kristin A. Eckert, Bradley D. Preston, Margaret S. Yoon, Stacy Washington, Alexander M. Chagovetz, Shu-Xia Li |
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| Rok vydání: | 1997 |
| Předmět: |
DNA Replication
Multidisciplinary biology DNA polymerase DNA repair viruses DNA polymerase II Genetic Complementation Test DNA replication Processivity Biological Sciences DNA Polymerase I Molecular biology DNA polymerase delta Recombinant Proteins Rats Phenotype Mutagenesis Escherichia coli biology.protein Animals Tyrosine Selection Genetic DNA polymerase I Polymerase |
| Zdroj: | Proceedings of the National Academy of Sciences. 94:1321-1326 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.94.4.1321 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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