DNA mismatch repair is a primary regulator of mutation production. The pathway is well known for its multiple mutation-avoidance functions, including the correction of DNA biosynthetic errors, inhibition of recombination between quasi-homologous sequences, and participation in the cellular response to DNA damage. Inactivation of human mismatch repair is the cause of Lynch syndrome, the most common hereditary cancer. Mismatch repair defects also have implications for cancer treatment because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs, a consequence of its function in the DNA damage response. Surprisingly, components of the mismatch repair system are also required for the generation of certain mutations including expanded triplet repeat sequences which are the cause of a number of neurodegenerative diseases. We are interested in the molecular mechanisms responsible for mismatch repair function in the control of mutation production. To this end we propose continuation of four ongoing lines of work: (1) MutL? interaction with MutS? is believed to play an important role in the initiation of mismatch repair, while interaction of the PCNA replication clamp with MutL? is required for activation and strand direction of the MutL? endonuclease. We will establish the molecular stoichiometries of these complexes and pursue further clarification of their nature. In the case of the PCNA*MutL? complex, we will also attempt to identify MutL? motif(s) required for the interaction. This aspect of the project will include evaluation of the role of zinc in Mut? endonuclease function. (2) The somatic expansion stage of (CAG)n/(CTG)n neurodegenerative diseases, which depends on the mismatch repair activities MutS?, MutL? and MutL?, can occur in post-mitotic cells. We have found that small CAG or CTG extrusions in covalently continuous DNA can serve as noncanonical PCNA loading sites, thus resulting in MutS?-dependent activation of MutL? endonuclease, which provides a simple mechanism for repair activation on non-replicating DNA. We are extending these studies to the analysis of (CAG)n/(CTG)n repeat processing by the human mismatch repair system in both extract and purified systems. Parallel studies will address the instability of expanded (GAA)n/(TTC)n Friedreich's ataxia disease alleles, which are subject to double strand cleavage in a MutS?? and MutL? endonuclease-dependent manner. (3) Mismatch repair function is required for checkpoint and apoptotic responses to O6-methylguanine, the cytotoxic lesion produced by SN1 DNA methylators. Heteroduplex DNA that contains O6-methylguanine on the template strand supports iterative cycles of abortive excision and repair that continues for several hours in cell extracts. We will attempt to clarify the nature of this reaction. (4) Ongoing collaborative studies with the laboratoy of Lorena Beese will address structural features of DNA processing by the human mismatch repair system with emphasis on the PCNA*MutL? complex.
DNA mismatch repair provides multiple mutation avoidance functions, and its inactivation is the cause of the most common hereditary cancer syndrome. Surprisingly, action of the pathway is also required for the production of certain mutations, including expanded triplet repeat sequences, the primary cause of a number of neurodegenerative disorders. By clarifying the molecular nature of mismatch repair, we hope to understand its functions in controlling the occurrence of mutation.
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