REGULATION OF MISMATCH REPAIR IN A MODEL E COLI SYSTEM

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DESCRIPTION: The long-term goal of this project is to understand the regulatio of the mut S, mutL, and mut H DNA repair genes and the possible implications o this regulation to DNA repair, spontaneous and induced mutagenesis, and chromosome stability in a model E. coli system. In addition, constructs and molecular tools from these regulation studies will be applied to genetic approaches to address key unresolved issues in mismatch repair. This work is important, because a form of mismatch repair mediated by homologues of E. coli MutS and MutL is present in most organisms, including humans, where it plays fundamental roles in inheritance, variation, evolution, recombination, and the development of cancers. The following five Specific Aims will be completed in four-year period. (I) Biochemical and genetic approaches will be used to elucidate the mechanisms of negative control of MutS and MutH expression by th Hfq RNA chaperone and RpoS sigma factor in exponentially growing and stationary-phase (nongrowing) cells. (II) Genetic and molecular biological analyses will be carried out on the structure and regulation of mut L and mut in E. coli K-12 and mut S, mut L, and mut H in natural ("quick-change") isolates of E. coli and Salmonella that may regulate their methyl-directed-mismatch (MDM) repair capacity differently from E. coli K-12. (III) Molecular biological and physiological approaches will be used to investigate the mechanism of saturation of MDM repair by mutagens, such as 2-aminopurine, and defects in replicative proof-reading. This study will address the long-standing, central problem in DNA repair of the reversal of MD repair saturation by overexpression of MutL or MutH, but not MutS. (IV) in viv and in vitro heteroduplex repair assays and in vivo homeologous recombination measurements will be used to assess the implications of mut S, mut L and mut H regulation to mutagenesis and repair capacity in exponentially growing, stationary-phase, starved, and heat-shocked cells. (V) Powerful genetic method will be developed to understand the in vivo functions and interactions of the MutL protein, and eventually MutS, MutH, and UvrD, in MDM repair, starting wit the hypothesis that MutL acts as a matchmaker or interface with MutS and MutH. This work will provide fundamental new knowledge about an important repair pathway that has been clearly implicated in human colon and sporadic cancers.