Quantitative Assays of Mismatch Repair Activity

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The Ohio State University

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Defects in the human mismatch repair (MMR) genes are the cause of Lynch syndrome as well as 10-40% of sporadic colorectal, gastric, endometrial, ovarian, and upper urinary tract tumors. The MMR system recognizes and repairs polymerase misincorporation errors and functions as a sensor in DNA damage signaling. MMR-deficient cells have increased mutations from unrepaired errors which drive tumorigenesis, and the lack of a DNA damage response results in resistance to several common cancer chemotherapeutic drugs. Single-molecule methods have proven to be insightful to the MMR process, however, evaluating whether the mismatch repair proteins used in experimentation match wildtype activity is vital for accurate results in single-molecule studies. This project will develop an assay to evaluate MMR activity on a variety of mismatched substrates. Several constructs will be built as part of the project to test the efficiency of the assay and MMR proteins through ssDNA and dsDNA hybridization. Wild-type cell extracts will be isolated and mixed with the mismatched constructs to evaluate wild-type activity and optimized conditions. Cell extracts without MMR activity will be mixed with MMR proteins and assayed for complementation using gel electrophoresis.



Assay, mismatch repair