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A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C
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 sequenc...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740443/ https://www.ncbi.nlm.nih.gov/pubmed/23946885 http://dx.doi.org/10.3390/genes4020134 |
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author | Chiraniya, Ankita Finkelstein, Jeff O’Donnell, Mike Bloom, Linda B. |
author_facet | Chiraniya, Ankita Finkelstein, Jeff O’Donnell, Mike Bloom, Linda B. |
author_sort | Chiraniya, Ankita |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3740443 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-37404432013-08-12 A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C Chiraniya, Ankita Finkelstein, Jeff O’Donnell, Mike Bloom, Linda B. Genes (Basel) Article 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. MDPI 2013-03-26 /pmc/articles/PMC3740443/ /pubmed/23946885 http://dx.doi.org/10.3390/genes4020134 Text en © 2013 by the authors; licensee MDPI, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Chiraniya, Ankita Finkelstein, Jeff O’Donnell, Mike Bloom, Linda B. A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C |
title | A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C |
title_full | A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C |
title_fullStr | A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C |
title_full_unstemmed | A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C |
title_short | A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C |
title_sort | novel function for the conserved glutamate residue in the walker b motif of replication factor c |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740443/ https://www.ncbi.nlm.nih.gov/pubmed/23946885 http://dx.doi.org/10.3390/genes4020134 |
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