Enzymology of DNA replication and repair

Our current research porjects are focused on Clamps and Clamp Loader proteins and on Mismatch Repair proteins.

Clamps and clamp loaders

DNA polymerases employ circular sliding clamps to catalyze rapid, processive synthesis of DNA.  These proteins serve an essential function in DNA replication, repair, and recombination, and are utilized in other important cellular processes, including cell cycle regulation.  A circular clamp has to be loaded onto duplex DNA for use by polymerases and other proteins, and this function is performed by a clamp loader, a multi-protein complex, that uses ATP to fuel the reaction.

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Our group is interested in understanding exactly how these critical proteins work, and to this end we measure transient events in the reaction in real time. We use stopped-flow and quench-flow methods coupled with global analysis of data from a variety of experiments to resolve the reaction mechanism.  The kinetic scheme shown here reflects our current thinking about the S. cerevisiae RFC clamp loader mechanism for loading PCNA clamp onto DNA (Chen et al., 2009, J. Mol Biol 388, 431-442).

Mechanism of RFC-catalyzed PCNA loading on DNA

Mismatch repair proteins

Mismatch repair proteins correct base-pair mismatches and small insertion/deletion loops that arise in DNA from errors made during DNA synthesis. If left uncorrected, such errors are fixed into the genome; thus, defective mismatch repair results in elevated mutation rates and genome instability.  In humans, an impaired mismatch repair system predisposes cells to cancer (e.g., hereditary nonpolyposis colon cancer).  The DNA repair process begins with the discovery of errors by MutS protein (eukaryotic Msh2-Msh6 and Msh2-Msh3 complexes), which subsequently signals other proteins to initiate excision and re-synthesis of the error-containing strand in a reaction fueled by ATP.  MutS also recognizes lesions produced by DNA damaging agents, except in this case the discovery leads to cell cycle arrest and/or apoptosis.  Thus, defects in MutS and other mismatch repair proteins result in double jeopardy as the affected cells are more susceptible to carcinogenesis and less susceptible to cytotoxic chemotherapeutic agents.

MutS/Msh6 function in DNA mismatch repair

We are currently studying T. aquaticus MutS and S. cerevisiae Msh2-Msh6 in order to resolve the kinetic mechanism of these proteins as they search for errors/lesions in DNA and use ATP to initiate a cellular response.  The kinetic scheme shown here reflects our current thinking on the S. cerevisiae Msh2-Msh6 mechanism whereby once the protein encounters an error in DNA (by 3D contact or 1D sliding on DNA), it forms a long-lived complex at the site, allowing rapid exchange of ATP for ADP and conversion of Msh2-Msh6 into a conformation that signals repair.

MutS/Msh6 mechansm of action in mismatch repair

Funding: National Science Foundation, National Institutes of Health, Connecticut Department of Public Health

Current lab members: Miho Sakato, Yayan Zhou, Anushi Sharma, Shreya Sawant, Bo Song, Xiaoyu Yu.