SPECIFICITY OF MISMATCH REPAIR IN ESCHERICHIA COLI

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Errors of DNA replication are potentially a major source of spontaneous and drug-induced mutation. Repair systems exist to minimize such damage. The best understood is DNA adenine methylation (Dam)-directed mismatch repair in Escherichia coli. Among the proteins involved in this repair system, three (MutH, MutL and MutS) are of particular importance. These three proteins appear to form a complex in vivo with DNA containing a replication error. MutL and MutS protein homologs have been detected in other bacteria, yeast, mouse and man indicating that the same basic repair mechanism is used. This proposal seeks to define, in E. coli, how the MutS protein (a) interacts with DNA, (b) with other Mut proteins and (c) the role of its ATPase activity by using genetic methods coupled with biochemical assays. Dominant negative mutations in the mutS gene have been selected by a newfangled procedure and all the mutations mapped so far by DNA sequencing are located in the most evolutionarily conserved amino acids. The mutants were sorted into different classes by their ability to be complemented in trans by MutS+, MutH+ and MutL+. Purified MutS protein from representative mutants will be assayed in vitro for ability to hydrolyze ATP, bind to DNA and assemble to form a complex (MutH,L,S) capable of incising DNA. These biochemical assays will then allow correlations to be made between location of the mutation in the gene and functional activity of protein domains. To confirm the assignment of protein-protein interfaces, a genetic selection will be used to isolate compensatory mutations which suppress a defective target Mut protein. This proposal represents a way not only to increase our knowledge about the origin of spontaneous birth defects and chemical teratogenesis but also to understand in general how proteins assemble into functional complexes.