title: Characterisation of the nucleotide and DNA coeffector binding sites of the herpes simplex virus type 1 (HSV-1) encoded helicase-primase complex and UL9 origin binding protein.
abstract: Nucleotide and DNA coeffector substrate binding site characterizations were performed on two HSV-1 DNA helicases fulfilling different roles in DNA replication. Single ATP-binding sites were identified for helicase-primase and UL9 protein (Km(ATP) 0.62 mM and 0.54 mM, respectively). Analysis of structural requirements for DNA-dependent NTP hydrolysis revealed comparatively stringent requirements for helicase-primase in accommodating base-modified NTP analogs whereas the UL9 protein was much more permissive in this respect; neither enzyme was dependent on the ribose 2' or 3' hydroxyls for NTP hydrolysis. Both helicase-primase and UL9 protein ATPase activities were inhibited by ADP or GDP; this effect was competitive rather than allosteric. The enhancement of ATPase activity on a single stranded (ss) DNA substrate as opposed to double stranded (ds) DNA was much more marked for helicase-primase than for the UL9 protein (Km(dsDNA)/Km(ssDNA) 60 and 9, respectively). The triphosphates of the antiviral agents acyclovir and penciclovir were not effective substrates for either helicase-primase or UL9 protein.
authors.affiliation: SmithKline Beecham Pharmaceuticals, Harlow, Essex, U.K.
authors.forename: D L
authors.initials: DL
authors.lastname: Earnshaw
authors.forename: R L
authors.initials: RL
authors.lastname: Jarvest
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D043583:Nucleoside-Triphosphatase; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D003854:Deoxyribonucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases