Abstract
Abstract A methyl methanesulfonate sensitive mutant of Bacillus subtilis appears to lack the enzyme DNA polymerase I. The absence of polymerase I permits the detection of a second DNA polymerizing enzyme in the mutant. This second polymerase is inhibitable by sulfhydryl antagonists, and shows maximal activity on double-stranded DNA substrates with many single strand breaks. Poly[d(A-T)] is less efficiently used as a substrate, in contrast to the substrate preferences of polymerase I. Compared to the wild type, the mutant is more sensitive to ultraviolet and x-irradiation, and is inefficient in reactivation of ultraviolet-irradiated SP01 bacteriophage, but is normal in genetic recombination.
Highlights
MethodsSubtilis (strain SB112 tryC pheA) cells were treated with N-methyl-N-nitroso guanidine to 0.1% survival (5)
DNA substrates with many single strand breaks
Two laboratories have reported techniques using transformable Bacillus subtilis in which the product of DNA synthesis is semiconservative, but replication of the chromosome appears to be sequential as judged by genetic evidence (2, 3)
Summary
Subtilis (strain SB112 tryC pheA) cells were treated with N-methyl-N-nitroso guanidine to 0.1% survival (5). Survivors were allowed to segregate prior to plating on supplemented minimal media, and were replica plated to plates containing O.O47oj, MMS,’ to isolate sensitive mutants. T: National Institutes of Health Research Career Development Awardee. Mapping-Competent cells (6) were prepared by the method of Stewart (7). Transforming DNA was prepared by the method of Marmur (8). Initial mapping was carried out by the method of Ephrati-Elizur (9). SB566 (tryC, thy) germinating spores released DNA which transformed competent SB1066 (purA, tryC, h&B, poZA5) cells.
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