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A DNA helicase from Schizosaccharomyces pombe stimulated by single-stranded DNA-binding protein at low ATP
1Basic Research Center, Nucleic Acid Biochemistry, Samsung Biomedical Research Institute, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-230, Korea.
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.
Insights
Researchers isolated DNA helicase I from fission yeast, finding it functions best with assistance. The single-stranded DNA-binding protein (SpSSB) enhances its activity at low ATP levels by lowering the ATP Km for unwinding.
Area of Science:
- Molecular Biology
- Biochemistry
- Yeast Genetics
Background:
- DNA helicases are crucial enzymes for DNA replication, repair, and recombination.
- Schizosaccharomyces pombe is a model organism for studying eukaryotic cell biology.
Purpose of the Study:
- To isolate and characterize a novel DNA helicase from Schizosaccharomyces pombe.
- To investigate the enzyme's activity and its regulation by ATP and other proteins.
Main Methods:
- Isolation and purification of DNA helicase I using various biochemical techniques.
- Enzyme activity assays including DNA unwinding and ATPase activity.
- Characterization of enzyme properties such as molecular mass and sedimentation coefficient.
- Investigating the effect of ATP concentration and single-stranded DNA-binding protein (SpSSB) on enzyme activity.
Main Results:
- DNA helicase I was purified as a 95 kDa polypeptide, with native molecular mass indicating a monomeric structure.
- The enzyme exhibited DNA helicase and single-stranded DNA-dependent ATPase activities.
- DNA helicase I showed high ATP turnover but inefficient unwinding at low ATP concentrations.
- SpSSB significantly stimulated DNA unwinding at low ATP levels by lowering the Km for ATP, without affecting ATPase activity.
Conclusions:
- DNA helicase I is a monomeric enzyme from S. pombe with distinct unwinding and ATPase activities.
- The enzyme's efficiency is modulated by ATP concentration, with SpSSB playing a key regulatory role.
- SpSSB enhances DNA helicase I activity at low ATP by improving its affinity for ATP, crucial for cellular processes.