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Reference: Chen S, et al. (2009) Mechanism of ATP-driven PCNA clamp loading by S. cerevisiae RFC. J Mol Biol 388(3):431-42

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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 remains open, especially the mechanism by which ATP is utilized for the task. Here we describe pre-steady state analysis of ATP hydrolysis, PCNA clamp opening and DNA binding by S. cerevisiae 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 bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC ATP PCNA((open)) intermediate is ready for 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 multi-step mechanism of a eukaryotic clamp loader, and furthermore facilitates comparative analysis of loaders from diverse organisms.

Reference Type
Journal Article
Authors
Chen S, Levin MK, Sakato M, Zhou Y, Hingorani MM
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