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 A 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 restored 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 Km for ATP in the unwinding reaction, allowing the helicase to function efficiently at low ATP concentrations.
Park
J S
JS
Basic Research Center, Nucleic Acid Biochemistry, Samsung Biomedical Research Institute, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-230, Korea.
Choi
E
E
Lee
S H
SH
Lee
C
C
Seo
Y S
YS
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D051716:Replication Protein A; D012568:Schizosaccharomyces / Q000201:enzymology*
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051716:Replication Protein A; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027093:TraI protein, E coli; D004265:DNA Helicases