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A DNA Helicase From Schizosaccharomyces PombeStimulated By Single-stranded DNA-binding Protein At Low ATP Concentration*

J. Park, E. Choi, S. Lee, C. Lee, Y. S. Seo
Published 1997 · Biology, Medicine

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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 Å 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 K m for ATP in the unwinding reaction, allowing the helicase to function efficiently at low ATP concentrations.
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