Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue
Journal article, 2007

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-2oxophenothiazine (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.

REVERSE-TRANSCRIPTASE

I KLENOW FRAGMENT

STRUCTURAL BASIS

BETA

RESOLUTION

PAIRS

CRYSTAL-STRUCTURE

MECHANISM

KINETIC

FIDELITY

NUCLEOTIDE INCORPORATION

Author

Gudrun Stengel

Scripps Research Institute

J. P. Gill

Scripps Research Institute

Peter Sandin

Chalmers

Marcus Wilhelmsson

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Bo Albinsson

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Bengt Nordén

Chalmers, Chemical and Biological Engineering, Physical Chemistry

D. Millar

Scripps Research Institute

Biochemistry

0006-2960 (ISSN) 1520-4995 (eISSN)

Vol. 46 43 12289-12297

Subject Categories

Physical Chemistry

Biochemistry and Molecular Biology

DOI

10.1021/bi700755m

More information

Latest update

9/10/2018