title: Translocation of E. coli RecQ helicase on single-stranded DNA.
abstract: A member of the SF2 family of helicases, Escherichia coli RecQ, is involved in the recombination and repair of double-stranded DNA breaks and single-stranded DNA (ssDNA) gaps. Although the unwinding activity of this helicase has been studied biochemically, the mechanism of translocation remains unclear. To this end, using ssDNA of varying lengths, the steady-state ATP hydrolysis activity of RecQ was analyzed. We find that the rate of ATP hydrolysis increases with DNA length, reaching a maximum specific activity of 38 ± 2 ATP/RecQ/s. Analysis of the rate of ATP hydrolysis as a function of DNA length implies that the helicase has a processivity of 19 ± 6 nucleotides on ssDNA and that RecQ requires a minimal translocation site size of 10 ± 1 nucleotides. Using the T4 phage encoded gene 32 protein (G32P), which binds ssDNA cooperatively, to decrease the lengths of ssDNA gaps available for translocation, we observe a decrease in the rate of ATP hydrolysis activity that is related to lattice occupancy. Analysis of the activity in terms of the average gap sizes available to RecQ on the ssDNA coated with G32P indicates that RecQ translocates on ssDNA on average 46 ± 11 nucleotides before dissociating. Moreover, when bound to ssDNA, RecQ hydrolyzes ATP in a cooperative fashion, with a Hill coefficient of 2.1 ± 0.6, suggesting that at least a dimer is required for translocation on ssDNA. We present a kinetic model for translocation by RecQ on ssDNA based on this characterization.
authors.affiliation: Department of Microbiology, Department of Molecular and Cellular Biology, Graduate Group in Biophysics, University of California, Davis, Davis, California 95616-8665, United States.
authors.forename: Behzad
authors.initials: B
authors.lastname: Rad
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Kowalczykowski
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D053484:RecQ Helicases / Q000737:chemistry*
chemical_list: D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D053484:RecQ Helicases