A DNA Helicase from Schizosaccharomyces pombeStimulated by Single-stranded DNA-binding Protein at Low ATP Concentration

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 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. 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. The unwinding of double-stranded DNA is required to provide single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; RPA, replication protein A; SpRPA, S. pombe RPA; FPLC, fast performance liquid chromatography; ssc, single-stranded circular; DTT, dithiothreitol; BSA, bovine serum albumin; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-3-O-thiotriphosphate; AppNp, 5′-adenylyl imidodiphosphate; AppCp, adenosine 5′-(β,γ-methylene)triphosphate; NEM, N-ethylmaleimide. 1The abbreviations used are: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; RPA, replication protein A; SpRPA, S. pombe RPA; FPLC, fast performance liquid chromatography; ssc, single-stranded circular; DTT, dithiothreitol; BSA, bovine serum albumin; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-3-O-thiotriphosphate; AppNp, 5′-adenylyl imidodiphosphate; AppCp, adenosine 5′-(β,γ-methylene)triphosphate; NEM, N-ethylmaleimide. templates for DNA transactions such as those involved in recombination, repair, and replication. For this purpose, DNA helicases catalyze a DNA strand separation reaction by breaking hydrogen bonds that hold the DNA strands together. DNA helicases use energy derived from the hydrolysis of nucleoside triphosphates for bond breakage and translocation of the enzyme along the DNA duplex. The DNA-unwinding activity catalyzed by a helicase exhibits a specific directionality (5′ → 3′ or 3′ → 5′) with respect to the DNA strand to which the enzyme is bound and moves.DNA helicases are ubiquitous and have been isolated from a wide variety of sources from bacteria to humans as well as bacteriophages and animal viruses. The biochemical properties of a number of DNA helicases have been reviewed extensively (1Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (335) Google Scholar, 2Thömmes P. Hübscher U. Chromosoma ( Berl. ). 1992; 101: 467-473Crossref PubMed Scopus (48) Google Scholar, 3Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 4Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar, 5West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Since detection of the first DNA helicase (Escherichia coli DNA helicase I) (6Abdel-Monem M. Durwald H. Hoffmann-Berling H. Eur. J. Biochem. 1976; 65: 441-449Crossref PubMed Scopus (138) Google Scholar), more than 60 distinct DNA helicase activities have been described (3Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 4Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar). Most organisms contain multiple helicases; for example, at least 12 different DNA helicases have been identified in E. coli (7Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar). Eight distinct helicases have been identified in Saccharomyces cerevisiae (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google and more than from of those from and P. Hübscher U. Chromosoma ( Berl. ). 1992; 101: 467-473Crossref PubMed Scopus (48) Google Scholar, 4Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar, M. M. PubMed Scopus Google Scholar, S. J. Full Text PDF PubMed Google Scholar, N. Tuteja R. 1990; PubMed Scopus Google Scholar, J. J. Full Text PDF PubMed Google P. E. R. Hübscher U. J. 1992; Full Text PDF PubMed Google Scholar, M. J. M. J. Biochem. ( ). 1994; Scopus Google Scholar). have that DNA and helicases contain distinct (3Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, Scopus Google Scholar). The S. cerevisiae data base H. H. M. P. H. 1996; Scopus Google more than with such helicase the number of identified DNA helicases to as the by these are of such a number of DNA helicases in the variety and of DNA and the distinct for a DNA The in of specific helicases are well and biochemical are The well helicases are and J. R. J. Full Text PDF PubMed Google Scholar), U. S. PubMed Scopus Google Scholar), S.W. S. J. Full Text PDF PubMed Google Scholar), M. J. Full Text PDF PubMed Google Scholar), H. P. R. J. PubMed Scopus Google P. J. U. S. PubMed Scopus Google Scholar), M. J. U. S. PubMed Scopus Google Scholar, E. M. M. U. S. PubMed Scopus Google Scholar), and U. S. 86: PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar). In involved in and in have been to be DNA helicases and of R. S. P. PubMed Scopus Google Scholar, P. S. 1994; PubMed Scopus Google of the well helicases is that is a of a and distinct to its specific For example, the DNA helicase in with protein is involved in of DNA DNA for by to and is required for of helicase activity S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). is the the helicase from E. with the of the DNA and the helicase activity of with in replication S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google J. M. Cell. 1996; 86: Full Text Full Text PDF PubMed Scopus Google described the of DNA helicases from that stimulated by as J. J. Full Text PDF PubMed Google Scholar, J. J. Full Text PDF PubMed Google Scholar). is a single-stranded DNA-binding protein and was first as an of the cell-free DNA replication system P. J. U. S. PubMed Scopus Google Scholar, U. S. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). is required for DNA repair, and in P. Cell. 1994; PubMed Scopus Google Scholar). In in that with protein involved in DNA replication E. J. 1992; PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar), E. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and PubMed Scopus Google Scholar, R. J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, X. Cell. PubMed Scopus Google Scholar). These that these DNA helicases in of DNA in to the of a in an of role in was such DNA have to an DNA helicase from the fission pombe. S. pombe is to with respect to and be to of the in S. In this the and of a DNA helicase that is stimulated by the SpRPA at ATP this have described the and biochemical properties of a DNA DNA helicase from S. pombe. helicase which was stimulated by the of SpRPA and an ATP-regenerating is the first DNA helicase to be from fission to a from S. DNA helicase and isolated from J. J. Full Text PDF PubMed Google Scholar, J. J. Full Text PDF PubMed Google Scholar). was stimulated by SpRPA, DNA helicase I in this is not to be a of the helicases described DNA helicase I was by SpRPA at concentrations of ATP (<0.2 helicase and stimulated by at high ATP concentrations mm). helicase DNA helicase I not a not distinct of DNA helicase I is that is a DNA-dependent ATPase that ATP at a high rate In contrast, helicase and active and had lower ATP turnover and DNA helicases from that in the as the DNA helicase I DNA helicase A P. E. R. Hübscher U. J. 1992; Full Text PDF PubMed Google Scholar), DNA helicases I and S. J. Full Text PDF PubMed Google from ATPase M. J. M. J. Biochem. ( ). 1994; Scopus Google Scholar), and DNA helicases and from N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar). these DNA the DNA helicase A was the that was to be significantly stimulated by the this enzyme was not stimulated by or E. coli In was significantly in than the S. pombe are to are to the helicase A was not at low ATP concentrations for stimulation of of biochemical properties such as for as energy to for ATPase and molecular that the DNA helicase I in this is to be a of any of these 3′ → helicase I is also not to be to any cerevisiae DNA helicase described (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, P. S. S. U. S. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, U. S. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). The protein P. S. S. U. S. PubMed Scopus Google Scholar), helicases and (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google Scholar), the protein J. Biochem. PubMed Scopus Google Scholar), and DNA helicase J. Full Text PDF PubMed Google in the → 3′ that of the helicase activity the a DNA helicase identified by and biochemical a 3′ → its helicase activity a U. S. PubMed Scopus Google in to the of S. pombe DNA helicase to be ATPase J. Full Text PDF PubMed Google is to DNA helicase I described in this biochemical of ATPase have not been that the separation of SpRPA from DNA helicase I was and was a gradient sedimentation or gel filtration in the of levels of m to the or the sedimentation or not The of SpRPA in the helicase gradient I to the of low levels of SpRPA on DNA helicase I this used the glycerol gradient that is relatively of SpRPA and and in low levels of SpRPA at the low ATP concentration mm). the that the helicase be SpRPA, the ATP concentration is a These also suggest that the activity in the absence of SpRPA may be to SpRPA in the helicase DNA helicases have been to be by RPA, the by which unwinding is by which SpRPA stimulated DNA helicase I a SpRPA and DNA helicase I a that the helicase to function efficiently at concentrations of ATP example, of the K m for or SpRPA the helicase to the the of ATP required for unwinding by a nonproductive ATP hydrolysis example, translocation along These of SpRPA in the DNA helicase I are not The by which SpRPA DNA helicase I to be In to the to be by in DNA a that from S. cerevisiae is in by DNA P. U. S. 1996; PubMed Scopus Google Scholar). a function for in DNA the function of DNA helicase I is not with RPA, a protein in repair, recombination, and in that DNA helicase I be involved in any not of these DNA helicase I in the 3′ to on the strand the replication involved in DNA replication. of the polypeptide that is a S. and The of the and DNA for DNA helicase I is The of and DNA helicase I to is of with Both biochemical and the function of DNA helicase I in S. pombe. In to helicases and use S. pombe to are to DNA helicase The unwinding of double-stranded DNA is required to provide single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; RPA, replication protein A; SpRPA, S. pombe RPA; FPLC, fast performance liquid chromatography; ssc, single-stranded circular; DTT, dithiothreitol; BSA, bovine serum albumin; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-3-O-thiotriphosphate; AppNp, 5′-adenylyl imidodiphosphate; AppCp, adenosine 5′-(β,γ-methylene)triphosphate; NEM, N-ethylmaleimide. 1The abbreviations used are: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; RPA, replication protein A; SpRPA, S. pombe RPA; FPLC, fast performance liquid chromatography; ssc, single-stranded circular; DTT, dithiothreitol; BSA, bovine serum albumin; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-3-O-thiotriphosphate; AppNp, 5′-adenylyl imidodiphosphate; AppCp, adenosine 5′-(β,γ-methylene)triphosphate; NEM, N-ethylmaleimide. templates for DNA transactions such as those involved in recombination, repair, and replication. For this purpose, DNA helicases catalyze a DNA strand separation reaction by breaking hydrogen bonds that hold the DNA strands together. DNA helicases use energy derived from the hydrolysis of nucleoside triphosphates for bond breakage and translocation of the enzyme along the DNA duplex. The DNA-unwinding activity catalyzed by a helicase exhibits a specific directionality (5′ → 3′ or 3′ → 5′) with respect to the DNA strand to which the enzyme is bound and DNA helicases are ubiquitous and have been isolated from a wide variety of sources from bacteria to humans as well as bacteriophages and animal viruses. The biochemical properties of a number of DNA helicases have been reviewed extensively (1Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (335) Google Scholar, 2Thömmes P. Hübscher U. Chromosoma ( Berl. ). 1992; 101: 467-473Crossref PubMed Scopus (48) Google Scholar, 3Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 4Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar, 5West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Since detection of the first DNA helicase (Escherichia coli DNA helicase I) (6Abdel-Monem M. Durwald H. Hoffmann-Berling H. Eur. J. Biochem. 1976; 65: 441-449Crossref PubMed Scopus (138) Google Scholar), more than 60 distinct DNA helicase activities have been described (3Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 4Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar). Most organisms contain multiple helicases; for example, at least 12 different DNA helicases have been identified in E. coli (7Matson S.W. Bean D.W. George J.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (269) Google Scholar). Eight distinct helicases have been identified in Saccharomyces cerevisiae (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google and more than from of those from and P. Hübscher U. Chromosoma ( Berl. ). 1992; 101: 467-473Crossref PubMed Scopus (48) Google Scholar, 4Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar, M. M. PubMed Scopus Google Scholar, S. J. Full Text PDF PubMed Google Scholar, N. Tuteja R. 1990; PubMed Scopus Google Scholar, J. J. Full Text PDF PubMed Google P. E. R. Hübscher U. J. 1992; Full Text PDF PubMed Google Scholar, M. J. M. J. Biochem. ( ). 1994; Scopus Google Scholar). have that DNA and helicases contain distinct (3Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, Scopus Google Scholar). The S. cerevisiae data base H. H. M. P. H. 1996; Scopus Google more than with such helicase the number of identified DNA helicases to as the by these are The of such a number of DNA helicases in the variety and of DNA and the distinct for a DNA The in of specific helicases are well and biochemical are The well helicases are and J. R. J. Full Text PDF PubMed Google Scholar), U. S. PubMed Scopus Google Scholar), S.W. S. J. Full Text PDF PubMed Google Scholar), M. J. Full Text PDF PubMed Google Scholar), H. P. R. J. PubMed Scopus Google P. J. U. S. PubMed Scopus Google Scholar), M. J. U. S. PubMed Scopus Google Scholar, E. M. M. U. S. PubMed Scopus Google Scholar), and U. S. 86: PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar). In involved in and in have been to be DNA helicases and of R. S. P. PubMed Scopus Google Scholar, P. S. 1994; PubMed Scopus Google Scholar). A of the well helicases is that is a of a and distinct to its specific For example, the DNA helicase in with protein is involved in of DNA DNA for by to and is required for of helicase activity S.C. Cell. Full Text PDF PubMed Scopus Google Scholar). is the the helicase from E. with the of the DNA and the helicase activity of with in replication S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google J. M. Cell. 1996; 86: Full Text Full Text PDF PubMed Scopus Google Scholar). In described the of DNA helicases from that stimulated by as J. J. Full Text PDF PubMed Google Scholar, J. J. Full Text PDF PubMed Google Scholar). is a single-stranded DNA-binding protein and was first as an of the cell-free DNA replication system P. J. U. S. PubMed Scopus Google Scholar, U. S. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). is required for DNA repair, and in P. Cell. 1994; PubMed Scopus Google Scholar). In in that with protein involved in DNA replication E. J. 1992; PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar), E. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), and PubMed Scopus Google Scholar, R. J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Full Text Full Text PDF PubMed Scopus Google Scholar, X. Cell. PubMed Scopus Google Scholar). These that these DNA helicases in of DNA in to the of a in an of role in was such DNA have to an DNA helicase from the fission pombe. S. pombe is to with respect to and be to of the in S. In this the and of a DNA helicase that is stimulated by the SpRPA at ATP concentrations. this have described the and biochemical properties of a DNA DNA helicase from S. pombe. helicase which was stimulated by the of SpRPA and an ATP-regenerating is the first DNA helicase to be from fission to a from S. DNA helicase and isolated from J. J. Full Text PDF PubMed Google Scholar, J. J. Full Text PDF PubMed Google Scholar). was stimulated by SpRPA, DNA helicase I in this is not to be a of the helicases described DNA helicase I was by SpRPA at concentrations of ATP (<0.2 helicase and stimulated by at high ATP concentrations mm). helicase DNA helicase I not a not distinct of DNA helicase I is that is a DNA-dependent ATPase that ATP at a high rate In contrast, helicase and active and had lower ATP turnover and DNA helicases from that in the as the DNA helicase I DNA helicase A P. E. R. Hübscher U. J. 1992; Full Text PDF PubMed Google Scholar), DNA helicases I and S. J. Full Text PDF PubMed Google from ATPase M. J. M. J. Biochem. ( ). 1994; Scopus Google Scholar), and DNA helicases and from N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar). these DNA the DNA helicase A was the that was to be significantly stimulated by the this enzyme was not stimulated by or E. coli In was significantly in than the S. pombe are to are to the helicase A was not at low ATP concentrations for stimulation of of biochemical properties such as for as energy to for ATPase and molecular that the DNA helicase I in this is to be a of any of these 3′ → helicase I is also not to be to any cerevisiae DNA helicase described (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, P. S. S. U. S. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, U. S. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). The protein P. S. S. U. S. PubMed Scopus Google Scholar), helicases and (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google Scholar), the protein J. Biochem. PubMed Scopus Google Scholar), and DNA helicase J. Full Text PDF PubMed Google in the → 3′ that of the helicase activity the a DNA helicase identified by and biochemical a 3′ → its helicase activity a U. S. PubMed Scopus Google in to the of S. pombe DNA helicase to be ATPase J. Full Text PDF PubMed Google is to DNA helicase I described in this biochemical of ATPase have not been that the separation of SpRPA from DNA helicase I was and was a gradient sedimentation or gel filtration in the of levels of m to the or the sedimentation or not The of SpRPA in the helicase gradient I to the of low levels of SpRPA on DNA helicase I this used the glycerol gradient that is relatively of SpRPA and and in low levels of SpRPA at the low ATP concentration mm). the that the helicase be SpRPA, the ATP concentration is a These also suggest that the activity in the absence of SpRPA may be to SpRPA in the helicase DNA helicases have been to be by RPA, the by which unwinding is by which SpRPA stimulated DNA helicase I a SpRPA and DNA helicase I a that the helicase to function efficiently at concentrations of ATP example, of the K m for or SpRPA the helicase to the the of ATP required for unwinding by a nonproductive ATP hydrolysis example, translocation along These of SpRPA in the DNA helicase I are not The by which SpRPA DNA helicase I to be In to the to be by in DNA a that from S. cerevisiae is in by DNA P. U. S. 1996; PubMed Scopus Google Scholar). a function for in DNA the function of DNA helicase I is not with RPA, a protein in repair, recombination, and in that DNA helicase I be involved in any not of these DNA helicase I in the 3′ to on the strand the replication involved in DNA replication. of the polypeptide that is a S. and The of the and DNA for DNA helicase I is The of and DNA helicase I to is of with Both biochemical and the function of DNA helicase I in S. pombe. In to helicases and use S. pombe to are to DNA helicase In this have described the and biochemical properties of a DNA DNA helicase from S. pombe. helicase which was stimulated by the of SpRPA and an ATP-regenerating is the first DNA helicase to be from fission to a from S. DNA helicase and isolated from J. J. Full Text PDF PubMed Google Scholar, J. J. Full Text PDF PubMed Google Scholar). was stimulated by SpRPA, DNA helicase I in this is not to be a of the helicases described DNA helicase I was by SpRPA at concentrations of ATP (<0.2 helicase and stimulated by at high ATP concentrations mm). helicase DNA helicase I not a not distinct of DNA helicase I is that is a DNA-dependent ATPase that ATP at a high rate In contrast, helicase and active and had lower ATP turnover and DNA helicases from that in the as the DNA helicase I DNA helicase A P. E. R. Hübscher U. J. 1992; Full Text PDF PubMed Google Scholar), DNA helicases I and S. J. Full Text PDF PubMed Google from ATPase M. J. M. J. Biochem. ( ). 1994; Scopus Google Scholar), and DNA helicases and from N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar). these DNA the DNA helicase A was the that was to be significantly stimulated by the this enzyme was not stimulated by or E. coli In was significantly in than the S. pombe are to are to the helicase A was not at low ATP concentrations for stimulation of of biochemical properties such as for as energy to for ATPase and molecular that the DNA helicase I in this is to be a of any of these 3′ → DNA helicase I is also not to be to any cerevisiae DNA helicase described (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, P. S. S. U. S. PubMed Scopus Google Scholar, J. Full Text PDF PubMed Google Scholar, U. S. PubMed Scopus Google Scholar, J. Biochem. PubMed Scopus Google Scholar). The protein P. S. S. U. S. PubMed Scopus Google Scholar), helicases and (8Li X. Yoder B.L. Burgers P.M.J. Chromosoma ( Berl. ). 1992; 102: S93-S99Crossref PubMed Scopus Google Scholar), the protein J. Biochem. PubMed Scopus Google Scholar), and DNA helicase J. Full Text PDF PubMed Google in the → 3′ that of the helicase activity the a DNA helicase identified by and biochemical a 3′ → its helicase activity a U. S. PubMed Scopus Google in to the of S. pombe DNA helicase to be ATPase J. Full Text PDF PubMed Google is to DNA helicase I described in this biochemical of ATPase have not been that the separation of SpRPA from DNA helicase I was and was a gradient sedimentation or gel filtration in the of levels of m to the or the sedimentation or not The of SpRPA in the helicase gradient I to the of low levels of SpRPA on DNA helicase I this used the glycerol gradient that is relatively of SpRPA and and in low levels of SpRPA at the low ATP concentration mm). the that the helicase be SpRPA, the ATP concentration is a These also suggest that the activity in the absence of SpRPA may be to SpRPA in the helicase DNA helicases have been to be by RPA, the by which unwinding is by which SpRPA stimulated DNA helicase I a SpRPA and DNA helicase I a that the helicase to function efficiently at concentrations of ATP example, of the K m for or SpRPA the helicase to the the of ATP required for unwinding by a nonproductive ATP hydrolysis example, translocation along These of SpRPA in the DNA helicase I are not The by which SpRPA DNA helicase I to be In to the to be by in DNA a that from S. cerevisiae is in by DNA P. U. S. 1996; PubMed Scopus Google Scholar). a function for in DNA the function of DNA helicase I is not with RPA, a protein in repair, recombination, and in that DNA helicase I be involved in any not of these DNA helicase I in the 3′ to on the strand the replication involved in DNA replication. of the polypeptide that is a S. and The of the and DNA for DNA helicase I is The of and DNA helicase I to is of with Both biochemical and the function of DNA helicase I in S. pombe. In to helicases and use S. pombe to are to DNA helicase are to M. and J. for with E. coli the also J. J. S. S. H. S. and J. for of the