title: The primary and secondary translocase activities within E. coli RecBC helicase are tightly coupled to ATP hydrolysis by the RecB motor.
abstract: Escherichia coli RecBC, a rapid and processive DNA helicase with only a single ATPase motor (RecB), possesses two distinct single-stranded DNA (ssDNA) translocase activities that can operate on each strand of an unwound duplex DNA. Using a transient kinetic assay to detect phosphate release, we show that RecBC hydrolyzes the same amount of ATP when translocating along ssDNA using only its primary translocase (0.81±0.05ATP/nt), only its secondary translocase (1.12±0.06ATP/nt), or both translocases simultaneously (1.07±0.09ATP/nt). A mutation within RecB (Y803H) that slows the primary translocation rate of RecBC also slows the secondary translocation rate to the same extent. These results indicate that the ATPase activity of the single RecB motor drives both the primary and secondary RecBC translocases in a tightly coupled reaction. We further show that RecBC also hydrolyzes the same amount of ATP (0.95±0.08ATP/bp) while processively unwinding duplex DNA, suggesting that the large majority, possibly all, of the ATP hydrolyzed by RecBC during DNA unwinding is used to fuel ssDNA translocation rather than to facilitate base pair melting. A model for DNA unwinding is proposed based on these observations.
authors: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors: Colin G
authors: CG
authors: Wu
authors: Fuqian
authors: F
authors: Xie
authors: Timothy M
authors: TM
authors: Lohman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000378:metabolism*; D006868:Hydrolysis
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli