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

doi:10.1074/jbc.272.17.11487

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

Suisheng Zhang and
Frank Grosse^‡

From Abteilung Biochemie, Institut für Molekulare Biotechnologie, Postfach 100813, D-07708 Jena, Germany

^‡ To whom correspondence should be addressed. Tel.: 49-3641-656291; Fax: 49-3641-656288; E-mail: fgrosse{at}imb-jena.de

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.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grant Gr 895/5-2. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y10658[GenBank].
↵¹ The abbreviations used are:

NDH II

human nuclear DNA helicase II

hnRNP

heterogeneous nuclear ribonucleoprotein

PCR

polymerase chain reaction

aa

amino acid(s)

bp

base pair(s)

NTA

nitrolotriacetic acid

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

ssDNA

single-stranded DNA

ssRNA

single-stranded RNA

dsDNA

double-stranded DNA

dsRBD

double-stranded RNA binding domain.
- Received October 22, 1996.
- Revision received February 19, 1997.