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 id entify and characterize mammalian DNA polymerase beta mutants. In this complementation system, wild-type rat DNA polymerase beta replaces bo th the replicative and repair functions of DNA polymerase I in the Esc herichia coli recA718 pol412 double mutant; our 263 DNA polymerase bet a mutants replace E. coli polymerase I less efficiently or not at all. Of the 10 mutants that have been shown to contain DNA sequence altera tions, 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 phenotyp e contain amino acid residue alterations within a putative nucleotide binding site of DNA polymerase beta. This approach for the isolation a nd evaluation of mutants of a mammalian DNA polymerase in E. coli may ultimately lead to a better understanding of the mechanism of action o f this enzyme and to precisely defining its role in vertebrate cells.