The long term objective of this project is to understand the underlying mechanisms of mutagenesis, and to define the mutational pathways and repair systems operating in the living cell. Because of the relationship between mutagenesis and carcinogenesis, this project has a direct bearing on human health. Also, a number of genetic diseases have been shown to result from defects in different DNA repair systems.
The specific aims i nvolve detecting and characterizing different mutator and antimutator-genes, which result in increased or decreased levels, respectively, of spontaneous mutations. This approach uncovers new mutational pathways and repair systems. Four new mutators, mutY, mutM, mutA, and mutC, will be studied in detail, since they define at least three new error avoidance and repair pathways. Different derivatives of phage Mu will be employed to insert into mutA and mutC, facilitating the cloning and sequencing of these genes. Rapidly reverting mutations in the bgl system will be employed together with characterized mutations in lacZ, to identify mutants with lowered rates of spontaneous mutations. These antimutator strains will be subjected to genetic and biochemical analysis to uncover the nature of the defect and to determine the mutational pathway affected. The control of different error avoidance and repair pathways will be investigated by isolating and characterizing genetic fusions of the 1ac region to the mutY, mutM, mutA, and mutC genes, among others. These fusions permit the measurement of gene expression in response to different environmental stimuli, as well as facilitating the isolation of regulatory mutants with altered control of the mutation avoidance pathways. A set of such mutants can help define how different repair systems are controlled. Understanding how the cell arranges the control of all of this error avoidance systems and repair pathways is important for understanding the molecular basis of spontaneous mutation. Different engineered strains will be constructed to aid in the measurement of mutation rate and in the detection of new mutator and antimutator strains.
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