Biology and MB&B

Graduate Student Career Retreat 2008

Name:  Siying Chen                

   Lab:  Manju Hingorani [MB&B]

 Abstract

Examination of the ATPase and DNA Recognition Mechanisms of Two Clamp Loaders, S. cerevisiae Replication Factor C & E. coli g complex

Siying Chen, MB&B Department, Wesleyan University

          Processive replication is achieved by tethering the polymerase to the DNA template by a circular sliding clamp protein. Clamps are loaded onto primer-template DNA by clamp loaders in a reaction fueled by ATP hydrolysis. Clamp loaders are pentameric AAA+ family (ATPases associated with diverse cellular activities) protein complexes conserved across all domains of life (for example, E. coli g complex & S. cerevisiae or human Replication Factor C). Our laboratory is interested in studying clamp loaders from different organisms to compare & contrast the mechanisms by which these proteins catalyze clamp assembly on DNA.

      The mechanism by which the g complex utilizes ATP binding and hydrolysis to catalyze clamp assembly has been revealed by extensive studies. However little is known about the detailed mechanism of any other clamp loaders, particularly eukaryotic clamp loaders, such as S. cerevisiae Replication Factor C (RFC). Our analysis of RFC ATP binding indicates that RFC alone binds 3 ATP molecules, but in the presence of PCNA clamp and/or primer-template DNA, it binds 4-5 ATP molecules, suggesting a link between the extra ATP binding & formation of a complex between RFC, PCNA, and DNA. RFC alone hydrolyzes ATP at a limiting rate of 0.025 second^-1, which increases to 0.05 second^-1 in the presence of PCNA clamp. PCNA stimulates formation of an “active” RFC-ATP-PCNA complex within 2 seconds while it takes 5-7 seconds for an RFC-ATP binary complex alone to achieve the same “active” conformation. Binding of primer-template DNA to this “active” complex triggers rapid hydrolysis of 3 ATP molecules at a rate of ~40 second^-1. Double-stranded DNA has the same effect of triggering while single-stranded DNA does not. This suggests that RFC primarily “recognizes” the duplex portion of primer-template DNA for clamp assembly.

     According to studies of the E. coli g complex (1,2),  the release of b clamp onto primer-template DNA is coupled with the reaction that all 3 ATPs bound to g complex being hydrolyzed rapidly, which indicates similarity to the RFC ATPase mechanism. But unlike in the S. cerevisiae system, ds-DNA does not have this triggering effect, which led to the conclusion that g complex is unable to release b clamp onto ds-DNA. Based on our experimental results, we found out that ds-DNA is also able to trigger rapid ATP hydrolsysis and release of the clmap onto DNA by g complex, but only in the presence of very high concentration of ds-DNA.

     We concluded that double stranded portion of pt-DNA appears to be an essential element for clamp loader to recognize for clamp assembly. While primer template junction is the preferred substrate for clamp assembly by E. coli g complex, RFC does not have a preference between ds-DNA and pt-DNA for clamp assembly.