title: Domain structure of human nuclear DNA helicase II (RNA helicase A).
abstract: Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression system. Recombinant NDH II unwound both DNA and RNA. Limited tryptic digestion produced active helicases with molecular masses of 130 and 100 kDa. The 130-kDa helicase missed a glycine-rich domain (RGG-box) at the carboxyl terminus, while the 100-kDa form missed both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and its RGG-box. Hence, the dsRBDs and the RGG-box were dispensable for unwinding. On the other hand, the isolated DEXH core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH2 terminus to the helicase core. Hence, an active helicase required part of the NH2 terminus, the DEXH core, and a C-terminal extension of the core. Both dsRBDs and the RGG-box were bacterially expressed as glutathione S-transferase fusion proteins. The two dsRBDs had a strong affinity to double-stranded RNA and cooperated upon RNA binding, while the RGG-box bound preferentially to single-stranded DNA. A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II.
authors.affiliation: Abteilung Biochemie, Institut für Molekulare Biotechnologie, Postfach 100813, D-07708 Jena, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: F
authors.initials: F
authors.lastname: Grosse
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000201:enzymology
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D020365:RNA Helicases