Biochemical and structural tools using recombinant proteins provide valuable information regarding protein function. We study two mechanisms of DNA repair that are necessary for proper cellular function, namely base excision repair (BER) and mismatch repair (MMR). BER is involved in the repair of non-bulky lesions such as DNA base oxidation and abasic sites, while MMR is essential for the repair of mismatches, insertions, and deletions that arise during DNA replication. Human NEIL1, one of 11 DNA glycosylases, catalyzes the first step of the BER pathway and interacts with replication proteins such as the homotrimeric proliferating cell nuclear antigen (PCNA) and the homotetrameric mitochondrial single-stranded DNA binding protein (mtSSB). We isolated complexes formed between NEIL1 and these proteins using gel filtration chromatography and probed the interactions using small angle X-ray scattering methods. In both instances, NEIL1 appears to interact with monomeric PCNA and mtSSB, leading us to hypothesize that the repair enzyme can stall replication when an oxidized lesion is encountered. We also use functional assays and structural methods to evaluate variants of uncertain significance (VUSs) within the PMS2 gene, one of 4 essential MMR genes associated with Lynch syndrome (LS). LS is a hereditary cancer syndrome that causes an increased lifetime risk of developing malignancies. We have identified several germline VUSs within the ATPase domain of PMS2 using information from genetic screening. In the current study, we present our findings on 2 PMS2 variants, where one variant (deletion of amino acids 42 – 44) is likely pathogenic resulting in a protein with severely impaired function. The second variant (G207E) possesses near wild-type activity and is likely benign. Our overall mission is to help with the reclassification of PMS2 VUSs to assist with proper medical management decisions.

Structural and biochemical studies to assess protein interactions and classify VUSs

2019 Spring National Meeting

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