TY - JOUR
T1 - Mechanism of ATP-Driven PCNA Clamp Loading by S. cerevisiae RFC
AU - Chen, Siying
AU - Levin, Mikhail K.
AU - Sakato, Miho
AU - Zhou, Yayan
AU - Hingorani, Manju M.
N1 - Funding Information:
This work was supported by National Institutes of Health grant GM64514-01 to M.M.H. We thank Stephen J. Benkovic and Michael O'Donnell for clones, Smita S. Patel for helpful discussions, and Carol Teschke for use of the Applied Photophysics stopped-flow instrument.
PY - 2009/5/8
Y1 - 2009/5/8
N2 - 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.
AB - 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.
KW - ATPase kinetics
KW - PCNA clamp
KW - RFC clamp loader
KW - processive DNA replication
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U2 - 10.1016/j.jmb.2009.03.014
DO - 10.1016/j.jmb.2009.03.014
M3 - Article
C2 - 19285992
AN - SCOPUS:64649100384
SN - 0022-2836
VL - 388
SP - 431
EP - 442
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -