Human cells possess a strand-specific mismatch repair system that is a homolog of the bacterial methyl-directed pathway as judged by similarities in specificity and mechanism. Like the bacterial system, human strand-specific repair contributes to genetic stability since several type of hypermutable human cell are deficient in the reaction. Such mutator cells include certain mutants that are tolerant to the cytotoxic action of DNA alkylating agents, with a second class defined by genetically unstable, RER+ (replication error prone) tumor cells such as those that occur in individuals with heritable nonpolyposis colon cancer (HNPCC). In this application we propose work along two major lines, the first of which will extend our study of mutator cell lines like those mentioned above. In collaboration with the laboratory of Dr. Bert Vogelstein, we will expand our analysis of RER+ tumor cell lines, with the objectives of this study being to assess generality of the association of mismatch deficiency with the RER+ phenotype and to classify repair-defective lines based on in vitro complementation. The other facet of the mutator cell work will involve test of RER+ tumor lines for tolerance to killing by alkylating agents, and in a collaborative study with Dr. Henry Friedman, test for mismatch repair defects in clinically derived tumor cells that have developed tolerance to chemotherapeutic DNA methylating agents. The second major line of work, which will be conducted in parallel with the mutator cell work, will involve fractionation of the human repair system, with the ultimate goals being reconstitution of the reaction in a purified system and elucidation of its molecular mechanism. Using biochemical methods and complementation of nuclear extracts derived from RER+ and alkylation- tolerant mutant cells, we have identified six (possibly seven) distinct activities required for the reaction. We intend to pursue isolation of these six components and any other required activities that may be identified during the course of the study, focusing initially on those components that are lacking in mutant cell lines.
Our aim i n this work will be to obtain near homogeneous preparations of each activity. In addition to their utility in addressing questions of mechanism, availability of purified components should facilitate identification of novel genetic loci associated with HNPCC. Moreover, the proposed mechanistic analysis may yield information that will prove useful in the development of biochemical diagnostics for this inherited disease.
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