| Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I. | |
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MedLine Citation:
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PMID: 22650319 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule Förster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations--open, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3'-5' exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair. |
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Authors:
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Svitlana Y Berezhna; Joshua P Gill; Rajan Lamichhane; David P Millar |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2012-06-29 |
Journal Detail:
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Title: Journal of the American Chemical Society Volume: 134 ISSN: 1520-5126 ISO Abbreviation: J. Am. Chem. Soc. Publication Date: 2012 Jul |
Date Detail:
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Created Date: 2012-07-11 Completed Date: 2012-11-05 Revised Date: 2013-04-16 |
Medline Journal Info:
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Nlm Unique ID: 7503056 Medline TA: J Am Chem Soc Country: United States |
Other Details:
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Languages: eng Pagination: 11261-8 Citation Subset: IM |
Affiliation:
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Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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DNA Polymerase I
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chemistry,
metabolism* Escherichia coli / chemistry, enzymology*, metabolism Fluorescence Resonance Energy Transfer* Models, Molecular Nucleotides / metabolism Protein Conformation Substrate Specificity |
| Grant Support | |
ID/Acronym/Agency:
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GM044060/GM/NIGMS NIH HHS; R01 GM044060/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Nucleotides; EC 2.7.7.-/DNA Polymerase I |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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