Domain Structure of Human Nuclear DNA Helicase II (RNA Helicase A)

• Published in: The Journal of biological chemistry 1997-04, Vol.272 (17), p.11487-11494
• Main Authors: Zhang, S, Grosse, F
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Baculoviridae - genetics; Cattle; Cell Nucleus - enzymology; Cloning, Molecular; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Helicases - metabolism; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - metabolism; Humans; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Peptide Fragments - metabolism; Protein Conformation; Recombinant Proteins - metabolism; RNA Helicases; RNA Nucleotidyltransferases - chemistry; RNA Nucleotidyltransferases - genetics; RNA Nucleotidyltransferases - metabolism; RNA, Double-Stranded - metabolism; RNA-Binding Proteins - metabolism; Sequence Analysis, DNA; T-Lymphocytes - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.17.11487

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 DE X H core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH 2 terminus to the helicase core. Hence, an active helicase required part of the NH 2 terminus, the DE X H 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.