Conformational Dynamics of DNA Polymerase Probed with a Novel Fluorescent DNA Base Analogue
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- 作者：Gudrun Stengel ; Joshua P. Gill ; Peter Sandin ; L. Marcus Wilhelmsson ; Bo Albinsson ; Bengt Nordé ; n ; David Millar
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：October 30, 2007
- 年：2007
- 卷：46
- 期：43
- 页码：12289 - 12297
- 全文大小：343K
- 年卷期：v.46,no.43(October 30, 2007)
- ISSN：1520-4995
文摘
DNA polymerases discriminate between correct and incorrect nucleotide substrates during a"nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotideincorporation. Despite the importance of this process in polymerase fidelity, the precise nature of themolecular events involved remains unknown. Here we report a fluorescence resonance energy transfer(FRET) system that monitors conformational changes of a polymerase-DNA complex during selectionand 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 wasincorporated 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 thefingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantlyfollowing binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingershad closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated thatthe DNA was more tightly bound by the polymerase under these conditions, consistent with the formationof a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured instopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measuredin rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step inthe overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, theseresults indicate that the FRET system can be used to probe enzyme conformational changes that arelinked to the biochemical function of DNA polymerase.