Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC

Siying Chen, Mikhail K. Levin, Miho Sakato, Yayan Zhou, Manju M. Hingorani

Research output: Contribution to journalArticle

27 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)431-442
Number of pages12
JournalJournal of Molecular Biology
Volume388
Issue number3
DOIs
StatePublished - May 8 2009

Keywords

  • ATPase kinetics
  • PCNA clamp
  • RFC clamp loader
  • processive DNA replication

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Chen, S., Levin, M. K., Sakato, M., Zhou, Y., & Hingorani, M. M. (2009). Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC. Journal of Molecular Biology, 388(3), 431-442. https://doi.org/10.1016/j.jmb.2009.03.014