title: The SF1 helicase encoded by the archaeal plasmid pTN2 of Thermococcus nautili.
abstract: We expressed, purified, and characterized the helicase encoded by ORF1 of the Thermococcus nautili pTN2 plasmid (Soler et al. Nucl Acids Res 38, 5088-5104, 2010). The enzyme, which belongs to the SF1 family of helicases, possesses NTPase activity, with a strong preference for ATP and GTP as compared to CTP and TTP; dATP was also a substrate. Triphosphatase activity was strongly stimulated by single-stranded DNA and, to a lesser extent, by double-stranded DNA. Unwinding of duplexes comprising a fluorescent oligonucleotide was monitored by fluorescence polarization spectroscopy and by polyacrylamide gel electrophoresis. As observed for enzymes of the same family, pTN2 helicase displays a strong preference for duplexes comprising a 3' single-stranded extension and proceeds from the 3' to the 5' end of the loading strand. Under the conditions of the in vitro assay, pTN2 helicase did not appear to be recycled, but stayed bound to single-stranded DNA, which explains why high concentrations of enzyme are required to unwind long stretches of duplex DNA. The helicase enhances the synthesis of double-stranded DNA by pTN2 primase and by T. nautili PolB polymerase primed by pTN2 primase but it did not enhance synthesis by Taq DNA polymerase.
authors.affiliation: Unité Biologie moléculaire du Gène chez les Extrêmophiles, Département de Microbiologie, Institut Pasteur, 25 rue de Dr Roux, 75724, Paris Cedex 15, France, pierre.beguin@pasteur.fr.
authors.forename: Pierre
authors.initials: P
authors.lastname: Béguin
authors.forename: Bruno
authors.initials: B
authors.lastname: Baron
authors.forename: Sukhvinder
authors.initials: S
authors.lastname: Gill
authors.forename: Nicole
authors.initials: N
authors.lastname: Charpin
authors.forename: Patrick
authors.initials: P
authors.lastname: Forterre
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D004265:DNA Helicases