Mutations in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer in humans. Transgenic mice in which the MMR genes have been inactivated are prone to cancer. However, the different knock-out mice develop different types of tumors, suggesting that the respective proteins may have non-overlapping roles in the preservation of genomic integrity and in tumor suppression. The principal investigator has developed a transgenic mouse assay system to study mutagenesis in vivo using a chromosomally-integrated, recoverable lambda phage shuttle vector carrying the bacterial supF gene as a mutation reporter. The broad, long-term objective of this proposal is to combine this in vivo mutation assay with the MMR-deficient animals to probe critical issues relating to genetic instability, carcinogenesis, and response to cancer therapy in the setting of MMR deficiency.
The specific aims are: 1) To generate second-generation transgenic mice with an improved supF reporter construct that is transcribed in mouse cells and that is modified to contain a signature sequence to identify sibling vs. independent mutations. 2) To examine spontaneous mutagenesis in hybrid mice, all carrying the XsupF shuttle vector, plus various combinations of alleles at the MMR loci, including PMS1, PMS2, MLH1, and MSH2. Each genotype will be compared for tissue-specific, age-related, and developmental differences. Mutation frequencies and spectra will be analyzed. 3) To examine induced mutagenesis in the mice by alkylating agents, UV light and base analogs in order to probe the role(s) of the MMR factors in cellular pathways other than mismatch correction, such as transcription-coupled repair. 4)To investigate the role of MMR in response to ionizing radiation, with emphasis on mutagenesis, cytotoxicity, and recognition of x-ray damage, based on the principal investigator`s preliminary data showing that MMR-deficient cells are x-ray resistant. 5) To test the role of MMR in suppressing genetic instability secondary to errors made by polymerase during base excision repair. Use will be made of transgenic mice with a targeted disruption of the polymerase locus, as well as the availability of a mutator variant of the polymerase to test the influence of MMR on polymerase -mediated mutagenesis.
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