Circular clamps are evolutionarily conserved ring-shaped proteins that play a central role in a range of cellular processes, including DNA replication and repair, chromosome structure maintenance and cell cycle regulation. Clamps bind a numerous proteins and influence the course of multiple interconnected pathways of DNA metabolism and cell cycle progression. It follows that investigation of the structure-function properties and mechanisms of action of these proteins is of considerable significance. Key to understanding how clamps work is an understanding of how they are loaded onto DNA and how they interact with and modulate the activities of target proteins from this position. The proposal focuses on a clamp from a eukaryotic model organism, S. cerevisiae PCNA, in order to tackle these fundamental questions. The plan is to investigate the mechanism of action of S. cerevisiae Replication Factor C clamp loader (RFC), a multi-protein complex that binds PCNA and DNA and creates a topological link between the two in an ATP-dependent reaction. With respect to a PCNA target, we plan to investigate the mechanism of action of S. cerevisiae Msh2-Msh6, an essential DNA mismatch repair protein that detects base pair errors in DNA and initiates repair in a an ATP-dependent reaction. There are leading crystal structures available for each of these proteins in different model systems;however, since clamps exert their effects via multiple transient contacts and conformational changes, the use of kinetic methods is essential to monitor the reactions and determine if clamps alter the order of events and/or rate constants governing the reaction mechanisms. Our recent pre-steady state kinetic analysis of S. cerevisiae RFC ATPase activity indicates rate-limiting step(s) likely associated with conformational changes in PCNA. This is an exciting discovery, which, if confirmed, would reveal a means by which clamps could control the activities of their protein targets. To this end, we propose to directly measure the kinetics and energetics of PCNA interactions with RFC and DNA and explore the structure-function properties underlying its actions during clamp loading. In complementary research, we propose to build on our studies of S. cerevisiae Msh2-Msh6 and address hypotheses currently under debate on how PCNA influences early steps in DNA mismatch repair. Having recently developed rapid kinetic methods that measure Msh2-Msh6 interactions with mismatched DNA and the coupled ATPase activity, we can explicitly determine whether and how PCNA modulates Msh2-Msh6 binding to DNA and initiation of mismatch repair. The proposed research will be conducted by graduate and undergraduate students in the laboratory. The findings will yield significant new knowledge about the workings of a eukaryotic clamp and clamp loader, and, importantly, about the workings of Msh2-Msh6, whose malfunction is associated with hereditary non-polyposis colon cancer and sporadic tumors in humans.
Understanding the mechanisms of action of circular clamps is integral to understanding the central role of these proteins in several interconnected processes of DNA metabolism and cell cycle regulation. The proposed research will utilize transient kinetics approaches to study the impact of the eukaryotic clamp PCNA on the workings of two essential protein complexes: DNA replication protein RFC, and DNA repair protein Msh2- Msh6. Knowledge gained from this study will help elucidate how clamps help regulate the critical processes of DNA replication and DNA mismatch repair.
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