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Genes 2013, 4(2), 134-151; doi:10.3390/genes4020134

A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C

1
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA
2
Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA
*
Author to whom correspondence should be addressed.
Received: 8 January 2013 / Revised: 19 March 2013 / Accepted: 20 March 2013 / Published: 26 March 2013
(This article belongs to the Special Issue DNA Replication)
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Abstract

In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC. View Full-Text
Keywords: DNA replication; clamp loader; AAA+ ATPase; Walker B motif; sliding clamp; ATP hydrolysis DNA replication; clamp loader; AAA+ ATPase; Walker B motif; sliding clamp; ATP hydrolysis
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Chiraniya, A.; Finkelstein, J.; O'Donnell, M.; Bloom, L.B. A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C. Genes 2013, 4, 134-151.

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