Title : A Novel Approach to the Biochemical Analysis of Variants in the Essential DNA Mismatch Repair Protein MLH 1

Approved: ________________________________ _ _________________________ Andrew Buermeyer Genomic integrity is crucial for the viability and function of a cell. One key pathway that acts to maintain genomic integrity is DNA mismatch repair ( MMR). MMR acts to correct base pair mismatches hat have escaped proofreading during DNA replication. The process of MMR is dependent on the protein heterodimer MutL α, composed of the proteins MLH1 and PMS2. Mutations in MLH1 are linked to a condition known a s Lynch syndrome, which is characterized by a predisposition to early-onset colorectal cance r. B cause MLH1 is a frequent target of mutations that disable MMR and therefore cause Lync h syndrome, research on mutations in MLH1 is of significant interest. Other laboratories have used an in vitro approach to study MMR, however these studies generally were not quant itative and too labor intensive for the analysis of multiple variants of MLH1. We present p reliminary data detailing a novel approach to the biochemical analysis of DNA mismatch repair. In our assay, MutL α is prepared from MLH1 and PMS2-deficient cells transfected to expres s wild type PMS2 and wild type or mutant MLH1. This recombinant MutL α is then used to complement a cellular extract from cells deficient in MLH1 and PMS2, reconstituting a comple te repair system. Our findings demonstrate that such an approach is capable of supporting mism atch repair in vitro with sufficient precision and reproducibility to support the comparative anal ysis of multiple mutants of MLH1 and PMS2.