Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli.
We have designed and utilized a bacterial complementation system to identify and characterize mammalian DNA polymerase beta mutants. In this complementation system, wild-type rat DNA polymerase beta replaces both the replicative and repair functions of DNA polymerase I in the Escherichia coli recA718 polA12 double mutant; our 263 DNA polymerase beta mutants replace E. coli polymerase I less efficiently or not at all. Of the 10 mutants that have been shown to contain DNA sequence alterations, 2 exhibit a split phenotype with respect to complementation of the growth defect and methylmethanesulfonate sensitivity of the double mutant; one is a null mutant. The mutants possessing a split phenotype contain amino acid residue alterations within a putative nucleotide binding site of DNA polymerase beta. This approach for the isolation and evaluation of mutants of a mammalian DNA polymerase in E. coli may ultimately lead to a better understanding of the mechanism of action of this enzyme and to precisely defining its role in vertebrate cells.
Sweasy
J B
JB
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Loeb
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics*; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D051381:Rats
D009838:Oligodeoxyribonucleotides; D004256:DNA Polymerase I