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J Mol Biol. 2009 May 8;388(3):431-42. doi: 10.1016/j.jmb.2009.03.014. Epub 2009 Mar 13.

Mechanism of ATP-driven PCNA clamp loading by S. cerevisiae RFC.

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  • 1Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.

Abstract

Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA, especially the mechanism by which ATP is utilized for the task, remains open. Here we describe pre-steady-state analysis of ATP hydrolysis, proliferating cell nuclear antigen (PCNA) clamp opening, and DNA binding by Saccharomyces cerevisiae replication factor C (RFC), and present the first kinetic model of a eukaryotic clamp-loading reaction validated by global data analysis. ATP binding to multiple RFC subunits initiates a slow conformational change in the clamp loader, enabling it to bind and open PCNA and to bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC.ATP.PCNA((open)) intermediate is ready for the entry of DNA into the clamp. DNA binding commits RFC to ATP hydrolysis, which is followed by PCNA closure and PCNA.DNA release. This model enables quantitative understanding of the multistep mechanism of a eukaryotic clamp loader and furthermore facilitates comparative analysis of loaders from diverse organisms.

PMID:
19285992
[PubMed - indexed for MEDLINE]
PMCID:
PMC2700029
Free PMC Article

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