title: Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A.
abstract: Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.
authors.affiliation: Department of Pathology, University of Washington, Box 357705, Seattle, WA 98195-7705, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Shen
authors.forename: M D
authors.initials: MD
authors.lastname: Gray
authors.forename: J
authors.initials: J
authors.lastname: Oshima
authors.forename: L A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D017122:Bacteriophage T4; D016367:Baculoviridae / Q000235:genetics; D003597:Cytosine Nucleotides / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D004926:Escherichia coli; D005090:Exodeoxyribonucleases; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D053484:RecQ Helicases; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D014764:Viral Proteins / Q000494:pharmacology; D014898:Werner Syndrome / Q000201:enzymology*; D000071657:Werner Syndrome Helicase
chemical_list: D000227:Adenine Nucleotides; D003597:Cytosine Nucleotides; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase