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ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change

Toseland, Christopher P., Webb, Martin R (2013) ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change. The Journal of biological chemistry, 288 (35). pp. 25183-93. ISSN 1083-351X. (doi:10.1074/jbc.M113.484667) (KAR id:47849)

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http://dx.doi.org/10.1074/jbc.M113.484667

Abstract

The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5'-3' activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2'(3')-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity.

Item Type: Article
DOI/Identification number: 10.1074/jbc.M113.484667
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: Chris Toseland
Date Deposited: 07 Apr 2015 11:56 UTC
Last Modified: 16 Nov 2021 10:19 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/47849 (The current URI for this page, for reference purposes)
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