Abstract
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.
Highlights
The unwinding of double-stranded DNA is required to provide single-stranded DNA1 templates for DNA transactions such as those involved in recombination, repair, and replication
Purification of DNA Helicase I—To detect DNA helicase activities in crude extracts prepared from S. pombe, we used a DNA helicase assay developed by Matson et al [16] and Venkatesan et al [17]
We considered two possibilities. (i) The weak helicase activity observed at a low ATP concentration might be attributed to either the depletion of ATP by a potent ATPase activity of DNA helicase I or the inhibitory effects of ADP formed by the hydrolysis of ATP. (ii) SpRPA may block a reaction by which ATP hydrolysis is uncoupled from DNA unwinding
Summary
The unwinding of double-stranded DNA is required to provide single-stranded DNA (ssDNA) templates for DNA transactions such as those involved in recombination, repair, and replication. The number of identified DNA helicases should continue to grow as the proteins encoded by these open reading frames are studied biochemically. The presence of such a large number of DNA helicases in individual cells probably reflects the variety and complexity of DNA metabolic reactions and the distinct structural template requirements for a given DNA helicase. RuvA provides DNA binding specificity for RuvB by guiding it to Holliday junctions and is required for activation of RuvB helicase activity [26] Another example is the DnaB protein, the replicative helicase from E. coli. We described the isolation of two DNA helicases from human cells that were markedly stimulated by
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