Our objective is to study the mechanism of DNA mismatch repair and the fidelity of DNA replication in Escherichia coli. Elimination of replication errors is important for the maintenance of genetic stability. Mutator genes are involved in error avoidance during DNA replication or post-replication mismatch repair. The existence of a mismatch repair system has been postulated to correct replication errors and account for gene conversion, high negative interference and map expansion. A novel type of repair for A/G mismatches to restore C/G pairs have been identified. This type of mismatch repair is independent of DNA adenine methylation (dam) and host mutHLS gene functions and appears to be different from the other known methylation-independent pathways as judged by the requirement for known E. coli DNA repair gene products. The research for genes involved in this type of mismatch correction and characterization of their encoded products will shed light on the mechanism of this repair pathway. The excision tracts of this dam- and mutS-independent repair will be determined. The neighboring sequences of the transversion mismatches will be mutagenized and tested for their influence on the repair. Mismatch repair reactions will be reconstituted with purified proteins. The mutT mutants of E. coli increase unidirectional A-T to C-G transversions at three orders of magnitude higher than the wild type. An in vitro assay for the mutT gene product has been developed based on an amber reversion assay and will be used to purify the mutT gene product from an overproducer. The role of the MutT protein in mismatch repair and DNA replication will be investigated. The stage (nucleotide incorporation, proofreading of post-replication mismatch repair) of error avoidance at which the MutT protein acts will be determined. Antibodies against the MutT protein enable us to ask whether the MutT protein is a component of DNA polymerase III. Finally, our attention focuses on the high specificity of MutT protein for A-T to C-G transversions and the interaction of MutT protein and its suppressor. Because mutagenesis and carcinogenesis are closely related, the knowledge of genes which control mutagenesis will benefit cancer research.
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