Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Finding Faults in DNA Mismatch Repair: Kinetic analysis of MutS actions on DNA
DNA Mismatch Repair is an evolutionarily conserved process that corrects base pair mismatches and small insertion/deletion loops (IDL) generated during DNA replication and recombination. MutS proteins initiate DNA mismatch repair by recognizing such errors, and trigger a series of events that result in excision of the incorrect DNA strand and DNA re-synthesis. Our goal is to understand how the S. cerevisiae MutS homolog, Msh2-Msh6 recognizes mismatches/IDLs and signals DNA repair in a reaction fueled by ATP. Our approach is to measure the DNA binding and ATPase activities of Msh2-Msh6 under pre-steady state or single turnover conditions, and thus determine its mechanism of action.
Recent data reveal that Msh2-Msh6 scans DNA for errors and, surprisingly, pauses at not only base pair mismatches/IDLs but also at alternate sites that may be characterized by local distortions or increased flexibility in the double helix. A key difference is that the half-life of Msh2-Msh6 at a mismatch/IDL is about 40-fold longer than at an alternate site (t½= 20 sec at a G:T mismatch versus 0.5 sec at a 2-Aminopurine(2Ap):T base pair, respectively). ATP binds rapidly to Msh2-Msh6 trapped at the G:T mismatch, facilitating its interaction with proteins downstream in the repair pathway. We propose that Msh2-Msh6 makes weak initial contacts with base pairs when scanning for errors, and distinguishes bona fide mismatches/IDLs by forming long-lived complexes specifically at these sites. Stabilization of Msh2-Msh6·mismatch complexes allows ATP binding to the protein, which in turn initiates DNA repair.