This proposal describes approaches designed to characterize a putative link between translation and DNA replication. The investigator has obtained preliminary evidence that there is an inducible mutagenic response called translational stress-induced mutagenesis (TSM). He found that the mutA and mutC mutator genes have a base substitution in the anticodons of the glyV or glyW tRNA genes that allow the mutant tRNAs to decode aspartate codons as glycine. The mutator phenotype is constitutive and the investigator argues that the phenotype is not due to rare glycine substitutions in the epsilon proofreading subunit of DNA polymerase III. In addition, the phenotype is recA-dependent but does not require functional umuD/C genes. The working hypothesis is that the pathway is recA-dependent but SOS-independent and is a response to translational stress rather than a result of mistranslation itself. The epsilon protein could be involved but the critical events in mutA cells are upstream of the epsilon subunit. Dr. Humayun proposes to carry-out three sets of experiments to further characterize the TSM response. The first set of experiments will test the hypothesis that translational stress rather than a specific amino acid substitution in a particular protein(s) induces TSM and will define the range of translational stress conditions that induce TSM. The second set of experiments will determine if translational stress will alter DNA replication fidelity by comparing DNA replication activities in induced and uninduced cells in an in vitro system. The third project will characterize how the TSM response differs from the umuD/C-dependent SOS pathway.
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