title: ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change.
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.
authors.affiliation: From the MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom and; Institut für Zelluläre Physiologie and Center for NanoScience, Physiologisches Institut, Ludwig Maximilians Universität, Munich 80336, Germany.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Toseland
authors.affiliation: From the MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, United Kingdom and. Electronic address: mwebb@nimr.mrc.ac.uk.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D008274:Magnesium
keywords: ATPases; DNA Helicase; Enzyme Mechanisms; Fluorescence; Kinetics; Superfamily 1