Mechanisms of Mismatch Repair Mediated Cell Death After Alkylating Agent Exposure

Alkylating agents are highly mutagenic, teratogenic and carcinogenic DNA damaging compounds present in the environment due to natural processes, manufacturing, smoked and cured food products and second hand smoke. One DNA lesion caused by alkylating agents is O6-methylguanine. This lesion accounts for the majority of the mutagenic capacity of alkylating agents due to its ability to form mispairs with thymine. This mispair is recognized by the DNA mismatch repair (MMR) system, but is an irreparable mispair that instead triggers an apoptotic cell death response. This R00 proposal focuses on answering long-standing questions into the mechanisms of MMR-dependent cell death after alkylating agent exposure. The Goellner laboratory will use separation-of-function mutations in multiple MMR genes to systematically evaluate this process at a level of mechanistic detail not previously available. The Goellner laboratory will employee dual model systems, both Saccharomyces cerevisiae and human cell culture models will be used. This will provide the benefits of well-defined genetics, quick strain and mutation generation and screening abilities available by using the simple eukaryotic yeast system combined with the greater level of complexity and translation to human disease provided by doing targeted experiments in a human cell culture system. The goals of this proposal are 1) To test the requirements for the various mechanistic functions (ATPase activity, conformational changes, binding partner recruitment, endonuclease activity etc.) of the upstream steps of MMR in response to alkylating agents, including the mispair recognition complex, Msh2-Msh6, and the secondary licensing factor, Mlh1-Pms1. 2) To test whether the excision step of MMR, either through Exonuclease 1 dependent or independent mechanisms, is required for the cell death response to alkylating agents, and whether chromatin remodeling factors, recently identified as being involved in MMR during the K99 phase of this project, play a role. 3) To determine how the MMR proteins activate and interact with downstream effectors influencing G2/M arrest and apoptosis activation. This work will utilize multiple available yeast strains containing separation of function mutations previously identified during my postdoctoral work and by others, these studies in yeast will prioritize mutations to be translated into human cell culture systems in order to better comprehend the human health consequences of mutations in MMR genes and how they interact with environmental agents. Cutting edge super resolution imaging of mismatch repair intermediates and downstream modulators will complement these genetic and cell biology studies. Together this proposal will provide significant advancement in our understanding of how environmental alkylating agents interact with cellular pathways to influence human health outcomes.