Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
An Active Clamping Role for PCNA during Assembly and Function on DNA
Circular clamp proteins enable processive DNA replication by tethering polymerases to the primer-template during DNA replication. Clamps also bind to and coordinate the functions of several other proteins on DNA, and are therefore essential for many DNA metabolic reactions (1). Clamps are loaded onto DNA in an ATP-fueled reaction by multi-subunit AAA+ protein complexes known as Clamp Loaders. The mechanism of action of these proteins is under active investigation, given their important role in genomic DNA replication, repair and recombination (2). According to our current kinetic model of the S. cerevisiae RFC clamp loader, ATP binding activates RFC, allowing it to bind and open the PCNA clamp for entry of primer-template DNA; DNA binding to RFC triggers ATP hydrolysis, PCNA closure around DNA and release of the PCNA•DNA complex from RFC (3). The question we are addressing is whether PCNA plays an active role in clamp assembly. The specific hypothesis is that interactions between positively charged residues on the inside of the clamp and DNA help trigger clamp closure around DNA and catalytic turnover of RFC, and such a hypothesis is in resonace with the emergence of electrostatic interaction as an important component of DNA-protein recognition. In order to test this hypothesis, we have generated several PCNA mutants in which individual conserved amino acids have been substituted with Alanine. Data from transient kinetic analysis of key events during clamp assembly, including ATP-bound RFC• PCNA•DNA complex association and dissociation, PCNA opening and closing, as well as phosphate release, indicates that alteration of a single PCNA-DNA contact can alter rate-limiting steps in the reaction. Thus, the kinetic data support the hypothesis that PCNA is more than just a passive ring around DNA—it is an active contributor to the clamp assembly reaction mechanism and, by extension, possibly other DNA metabolic reactions as well.
This research is supported by funding from NSF and NIH.