I propose to examine the repair of mismatched nucleotides in Escherichia coli using purified heteroduplex plasmid DNA. The major goal of this study is to determine the enzymes involved in the repair of mismatched nucleotides generated during the process(es) of General Genetic Recombination and where the heteroduplex DNA is symmetrically methylated such as after mutagenesis. The repair of mismatched nucleotides is a complex process involving several overlapping responses. We have shown that there are at least three pathways that lead to the repair of mismatched nucleotides: i) the previously described dam Instructed Pathway which catalyzes co-repair of unmethylated and hemi-methylated substrates and requires the mutH, mutL, mutS, and uvrD gene products, ii) a pathway that leads to co-repair of intra-genic mismatches in fully methylated DNA and requires the mutS and uvrD gene products, and iii) a pathway that leads to the independent repair of intra-genic mismatches and requires the recF and recJ gene products. The co-repair of intra-genic mismatched nucleotides on fully methylated templates is the most efficient previously undescribed pathway for repair and therefore the most accessible to study. Strains containing mutations in recF have been shown to be deficient in genetic recombination and repair of environmentally induced damage and is also of interest to this proposal. The heteroduplex plasmid DNA substrates used in this study are designed to measure both co-repair and independent repair of mismatched nucleotides. Several lines of investigation will be used in this study: 1) analysis of the modes of heteroduplex DNA repair in vivo using differentially methylated substrates and combination of known mutations affecting repair, 2) analysis of new mutations and revertants of both the co-repair and independent-repair phenotypes selected after mutagenesis using a novel heteroduplex repair screening procedure, and 3) development of an in vivo assay system based on the repair of heteroduplex plasmid substrates in order to purify the protein component(s) of the methylation-independent repair pathway(s) using in vitro complimentation and reconstitution assays. The ultimate goal of this research is to reconstitute a mismatch repair system using purified proteins and to determine the mechanism of heteroduplex DNA repair and its relationship to mutagenesis and gene conversion. The techniques used in this study should be generally applicable.
