Book of Abstracts: Albany 2005
Defining the ATPase Mechanism of Replication Factor C, the S.cerevisiae Clamp Loader
DNA replication requires the collaborative efforts of a large number of proteins. The core protein DNA polymerase, which catalyzes phosphodiester bond formation, tends to dissociate from DNA after extending only 10-30 nucleotides. It requires a sliding clamp to tether it to the primer-template. In complex with the clamp, the polymerase can rapidly incorporate thousands of nucleotides into a growing polymer without dissociating from DNA. Sliding clamps are ring-shaped proteins with a central opening large enough to encircle duplex DNA. Clamps have to be loaded on primer-template DNA by another accessory protein called a clamp loader. Clamp loaders load the clamps onto primer-template DNA in a reaction fueled by ATP hydrolysis. They are conserved multi-subunit proteins widely employed in both prokaryotes & eukaryotes, e.g. γ complex in E. coli is a pentameric protein with 3 ATP binding sites. The mechanism by which the γ complex utilizes ATP binding and hydrolysis to catalyze clamp assembly has been studied extensively, however very little is known about the detailed mechanism of any other clamp loaders, particularly eukaryotic clamp loaders. Replication Factor C, the clamp loader in S. cerevisiae is a hetero-pentameric protein with 4 intact ATP binding sites. Our lab is interested in understanding how this protein uses ATP for clamp assembly on DNA.
We have over-expressed and purified S. cerevisiae RFC. RFC alone binds 2 ATP molecules. In the presence of PCNA clamp and/or primer-template DNA it binds 3-4 ATP molecules, suggesting a link between its ATP binding activity & PCNA/DNA binding activities. The steady state ATPase rate is also stimulated by PCNA and DNA. Most interestingly, using rapid kinetic analysis we have found that primer-template DNA and PCNA clamp binding to RFC specifically triggers rapid hydrolysis of all 4 ATPs bound to the clamp loader. We do not observe this activity with PCNA or primer-template DNA or with other DNA substrates that are known to interact with RFC. The implications of these ATPase kinetics for the RFC clamp loading mechanism will be discussed.
Molelular Biology & Biochemistry Department