title: Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA.
abstract: Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8231, Saint Louis, MO 63110-1093, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fischer
authors.forename: Nasib K
authors.initials: NK
authors.lastname: Maluf
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002232:Carbocyanines; D002384:Catalysis; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D019793:Fluorescein; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D009068:Movement; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D002232:Carbocyanines; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C089187:cyanine dye 3; D000255:Adenosine Triphosphate; D006493:Heparin; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases; D019793:Fluorescein