Abstract:
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 MutLα, composed of the proteins MLH1 and PMS2.
Mutations in MLH1 are linked to a condition known as Lynch syndrome, which is characterized
by a predisposition to early-onset colorectal cancer. Because MLH1 is a frequent target of
mutations that disable MMR and therefore cause Lynch 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 quantitative and too labor intensive for the
analysis of multiple variants of MLH1. We present preliminary data detailing a novel approach
to the biochemical analysis of DNA mismatch repair. In our assay, MutLα is prepared from
MLH1 and PMS2-deficient cells transfected to express wild type PMS2 and wild type or mutant
MLH1. This recombinant MutLα is then used to complement a cellular extract from cells
deficient in MLH1 and PMS2, reconstituting a complete repair system. Our findings demonstrate
that such an approach is capable of supporting mismatch repair in vitro with sufficient precision
and reproducibility to support the comparative analysis of multiple mutants of MLH1 and PMS2.
