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

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

1
, 2
, 2
 and 1,*
Received: 8 January 2013; in revised form: 19 March 2013 / Accepted: 20 March 2013 / Published: 26 March 2013
(This article belongs to the Special Issue DNA Replication)
View Full-Text   |   Download PDF [407 KB, uploaded 26 March 2013]
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.
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 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

AMA Style

Chiraniya A, Finkelstein J, O'Donnell M, Bloom LB. A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C. Genes. 2013; 4(2):134-151.

Chicago/Turabian Style

Chiraniya, Ankita; Finkelstein, Jeff; O'Donnell, Mike; Bloom, Linda B. 2013. "A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C." Genes 4, no. 2: 134-151.



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