Role of transcription in adaptive mutagenesis

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DESCRIPTION (provided by applicant): Altered cellular functions, originated by mutant proteins, may enhance the potential for pathogens to overcome host responses and antibiotic therapy. The main goal in this proposal is to characterize, at the molecular level, novel cellular mechanisms that generate mutant proteins as part of the stress-induced increase in genetic diversity. Such mechanisms have the potential for influencing processes as diverse as evolution and the development of neoplasms. Bacillus subitlis provides a readily accessible paradigm for the study of formation of mutant transcripts, the control of cell differentiation and stress induced mutagenesis. The specific hypothesis in this research is that the mfd gene in Bacillus subtilis contributes to the generation of mutated transcripts in non-growing cells. It is speculated that Mfd, a factor involved in repair of transcribed DNA, mediates the generation of mutations by increasing transcription and allowing the formation of mutated transcripts in the presence of DNA lesions. The specific aims are to investigate the effects of mfd on transcription in Bacillus subtilis, and involve two kinds of experiments: 1) We will investigate rate of transcription effects on stationary phase mutagenesis: We will manipulate levels of transcription, by inducible systems, in conditions of starvation/stress and measure transcription and accumulation of mutations in genes under selection. It is expected that high levels of transcription correlate with increases in frequency of mutation. 2) We will investigate transcriptional bypass in stationary phase: We will examine the generation of mutated transcripts in conditions of starvation/stress by using a firefly luciferase (luc)-reporter system recently developed. In this system, non-mutated messages generate no luciferase activity, whereas bypass or misreading by RNAP generates a functional message that could be detected in the absence of growth. Also, these experiments will determine the effect of this gene on these processes. This novel research will contribute to our understanding of genetic programs of cells under stress. The main goal of this work is to understand, at the molecular level, new processes that generate mutations when cells are in stress conditions. Such mechanisms have the potential to influence processes as diverse as evolution, the development of cancer and how pathogens overcome host responses and antibiotic therapy.