Genetic Requirements for Protection Against Bleomycin Toxicity

Bleomycin is known to cause double strand breaks in vitro. Little is known, however, about its mechanism of genotoxicity in vivo. One way to probe the mechanism of genotoxicity of a DNA damaging agent in vivo is to compare the relative sensitivities of a wild type Escherichia coli strain to a panel of isogenic repair deficient mutants. If the pathway defective in the mutant is known (e.g., base excision repair, alkyltransferase repair, nucleotide excision repair, and so on), the sensitivity of the mutant can reveal mechanistic insight into the mode of killing by the DNA damaging agent. In this study, mutants deficient in recombinational repair, specifically recF, recBCD, ruvABC, recG and recGruvC, were examined for sensitivity to bleomycin. This sensitivity was tested in both dividing and non-dividing cells in order to analyze the effect of cell division on the cytotoxicity of bleomycin. When non-dividing cells were treated, the recBCD and recGruvC mutants, demonstrated high sensitivity to bleomycin. The recF mutant, on the other hand, demonstrated no sensitivity. These results were consistent with the conclusion that 2 bleomycin induces double strand breaks in vivo that are repaired by the recombinational repair double strand break pathway. It also suggests that no damage was induced by bleomycin that required repair by the daughter strand gap pathway. Examining the sensitivity of recombinational repair deficient mutants to bleomycin also gave new insights about the mechanism of recombinational repair. Both ruvABC and recG gene products resolve Holliday junctions; however, they are thought to work on separate recombinational pathways. In this study, although the recGruvC strain was highly sensitive to bleomycin, the ruvABC and the individual recG and ruvC strains were not. This result suggested redundancy in the functions of the RecG and RuvABC proteins. Dividing cells showed a marked increase in sensitivity to bleomycin as compared to non-dividing cells. In addition, the functional redundancy of recG and ruvABC mutants was no longer seen. The ruvABC strain demonstrated high sensitivity equal to that of the recGruvC and recBCD strains whereas the recG and ruvC strains were only slightly sensitive. Under these conditions of increased cytotoxicity, additional functions of the RuvAB enzymes became important for suppression of toxicity. This result suggested a change in the mechanism of bleomycin's genotoxicity. Thesis Supervisor: John Essigmann Title: Professor of Chemistry and Toxicology