Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue.

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Research Abstract Details 

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Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Abstract Text:

Gudrun Stengel,Joshua P Gill,Peter Sandin,L Marcus Wilhelmsson,Bo Albinsson,Bengt ,David Millar,Gudrun Stengel,Joshua P Gill,Peter Sandin,L Marcus Wilhelmsson,Bo Albinsson,Bengt ,David Millar,

DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2-oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Publishing Authors By Initials

G Stengel,JP Gill,P Sandin,LM Wilhelmsson,B Albinsson,B ,D Millar,G Stengel,JP Gill,P Sandin,LM Wilhelmsson,B Albinsson,B ,D Millar,

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Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 46

Page Numbers: 12289-97

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 4

MONTH: 10

YEAR: 2007

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Information

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LANGUAGE: eng

NlmUniqueID: 370623

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Grant and Affiliation Information for Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue.

AFFILIATION: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM44060

ACRONYM: GM

MEDLINETA: Biochemistry

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