DNA Mismatch Repair Protein Interactions on Mismatch‐Containing DNA

Lynch syndrome (LS) is the most common form of hereditary colon cancer. Stemming from the inheritance of mutations in the DNA mismatch repair (MMR) genes, LS increases lifetime risk for colonic and certain extra‐colonic cancers. Unlike sporadic cancers, LS cancers have an average age of onset of 40 years‐old. MMR improves replication fidelity by recognizing and coordinating the repair of single base‐pair mismatches and small insertion/deletion loops. When deficient for MMR, cells acquire a mutator phenotype that increases the rate of mutation in other important oncogenes and tumor suppressors. This eventually leads to cancer development. The MMR pathway is composed of four main genes: MSH2 , MSH6 , MLH1 and PMS2 . MSH2 and MSH6 proteins form a heterodimer that recognizes mismatches on DNA and recruits a heterodimer of MLH1 and PMS2. Together, they signal for Exonuclease I to remove the erroneous region on the nascent strand. Previous studies have indicated that MSH2‐MSH6 bound to ATP forms a sliding clamp on the DNA that is required for its interaction with MLH1‐PMS2. However, the nature of this interaction for downstream signaling remains elusive. Current models propose that MSH2‐MSH6 and MLH1‐PMS2 form a quaternary structure on DNA and function as a unit to promote repair, but there is no direct evidence for this structure. We therefore explored the hypothesis that the two heterodimers form stable quaternary structures that function as a unit to signal excision. Using an in vitro system with biotinylated mismatch‐containing DNA and streptavidin coated magnetic beads, we isolated MMR protein‐DNA complexes and studied their interaction and stability. We confirmed that MSH2‐MSH6 can form sliding clamps on mismatch‐containing DNA in the presence of ATP. We next found that while initial loading of MLH1‐PMS2 on mismatch‐containing DNA requires these MSH2‐MSH6 sliding clamps, MLH1‐PMS2 remains stably bound to DNA even after MSH2‐MSH6 has dissociated. We also found that MSH2‐MSH6 is less stable on DNA in the presence of MLH1‐PMS2. These results suggest that MSH2‐MSH6 loads MLH1‐PMS2 onto mismatch‐containing DNA and that a stable, quaternary unit may not necessarily be required. These studies will improve our knowledge of the mechanism by which MMR maintains genomic stability and, ultimately, improve prevention and treatment of MMR‐defective cancers. Support or Funding Information Connecticut Institute for Clinical and Translational Science (CICATS)