A DNA helicase from Schizosaccharomyces pombe stimulated by single-stranded DNA-binding protein at low ATP concentration

A DNA helicase named DNA helicase I was isolated from cell-free extracts of the fission yeast Schizosaccharomyces pombe. Both DNA helicase and single-stranded DNA-dependent ATPase activities copurified with a polypeptide of 95 kDa on an SDS-polyacrylamide gel. The helicase possessed a sedimentation coefficient of 6.0 S and a Stokes radius of 44.8 Angstrom determined by glycerol gradient centrifugation and gel filtration analysis, respectively. From these data the native molecular mass was calculated to be 110 kDa, indicating that the active enzyme is a monomer. The DNA-unwinding and ATP hydrolysis activities associated with DNA helicase I have been examined. One notable property of the enzyme was its relatively high rate of ATP turnover (35-50 molecules of ATP hydrolyzed/s/enzyme molecule) that may contribute to its inefficient unwinding activity at low concentrations of ATP (<0.2 mM). Addition of an ATP-regenerating system to the reaction mixture re stored the DNA-unwinding activity of the enzyme. S. pombe single-stranded DNA-binding protein (SpSSB, also called SpRPA) stimulated the DNA helicase activity significantly at low levels of ATP (0.025-0.2 mM) even in the absence of an ATP-regenerating system. In contrast, SpRPA had no effect on ATP hydrolysis at any ATP concentration examined. These observations suggest that the stimulation of DNA unwinding by SpRPA is not simply a result of suppression of nonproductive ATP hydrolysis. Rather, the role of SpRPA is to lower the K-m for ATP in the unwinding reaction, allowing the helicase to function efficiently at low ATP concentrations.
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Issue Date
1997-07
Language
ENG
Keywords

SV40 T-ANTIGEN; REPLICATION PROTEIN; SACCHAROMYCES-CEREVISIAE; DEPENDENT ATPASE; CALF THYMUS; CELLS; YEAST; REPAIR; PURIFICATION; PRIMASE

Citation

JOURNAL OF BIOLOGICAL CHEMISTRY, v.272, no.30, pp.18910 - 18919

ISSN
0021-9258
DOI
10.1074/jbc.272.30.18910
URI
http://hdl.handle.net/10203/74839
Appears in Collection
BS-Journal Papers(저널논문)
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