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• W1966347623 abstract "Werner Syndrome (WS) is a human progeroid disorder characterized by genomic instability. The gene defective in WS encodes a 3′ → 5′ DNA helicase (Gray, M. D., Shen, J.-C., Kamath-Loeb, A. S., Blank, A., Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A.(1997) Nat. Genet. 17, 100–103). Sequence alignment analysis identified an N-terminal motif in WRN that is homologous to several exonucleases. Using combined molecular genetic, biochemical, and immunochemical approaches, we demonstrate that WRN also exhibits an integral DNA exonuclease activity. First, whereas wild-type recombinant WRN possesses both helicase and exonuclease activities, mutant WRN lacking the nuclease domain does not display exonucleolytic activity. In contrast, WRN proteins with defective helicase activity are active in exonucleolytic digestion of DNA. Second, the exonuclease co-purifies with the 160-kDa WRN protein and its associated DNA helicase and ATPase activities through successive steps of ion exchange and affinity chromatography, suggesting that all three activities are physically associated. Lastly, anti-WRN antiserum specifically co-precipitates the WRN helicase and exonuclease activities indicating that both activities reside on the same antigenic WRN polypeptide. The association of an exonuclease with WRN distinguishes it from other RecQ homologs and raises the possibility that the distinct phenotypic characteristics of WS may be due in part to a defective exonuclease. Werner Syndrome (WS) is a human progeroid disorder characterized by genomic instability. The gene defective in WS encodes a 3′ → 5′ DNA helicase (Gray, M. D., Shen, J.-C., Kamath-Loeb, A. S., Blank, A., Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A.(1997) Nat. Genet. 17, 100–103). Sequence alignment analysis identified an N-terminal motif in WRN that is homologous to several exonucleases. Using combined molecular genetic, biochemical, and immunochemical approaches, we demonstrate that WRN also exhibits an integral DNA exonuclease activity. First, whereas wild-type recombinant WRN possesses both helicase and exonuclease activities, mutant WRN lacking the nuclease domain does not display exonucleolytic activity. In contrast, WRN proteins with defective helicase activity are active in exonucleolytic digestion of DNA. Second, the exonuclease co-purifies with the 160-kDa WRN protein and its associated DNA helicase and ATPase activities through successive steps of ion exchange and affinity chromatography, suggesting that all three activities are physically associated. Lastly, anti-WRN antiserum specifically co-precipitates the WRN helicase and exonuclease activities indicating that both activities reside on the same antigenic WRN polypeptide. The association of an exonuclease with WRN distinguishes it from other RecQ homologs and raises the possibility that the distinct phenotypic characteristics of WS may be due in part to a defective exonuclease. Werner syndrome bovine serum albumin dithiothreitol polyacrylamide gel electrophoresis. Werner Syndrome (WS)1 is an inherited disease characterized by an early onset of atherosclerosis, osteoporosis, diabetes mellitus, and cancers of non-epithelial cell origin (1Epstein C.J. Martin G.M. Schultz A.L. Motulsky A.G. Medicine. 1966; 45: 177-221Crossref PubMed Scopus (735) Google Scholar, 2Goto M. Miller R.W. Ishikawa Y. Sugano H. Cancer Epidemiol. Biomark. Prev. 1996; 5: 239-246PubMed Google Scholar). Cultured cells from WS patients also present a shortened replicative lifespan (3Martin G.M. Sprague C.A. Epstein C.J. Lab. Invest. 1970; 23: 86-92PubMed Google Scholar) and increased genetic instability (4Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar). The genomic instability of WS cells is manifested at the cytogenetic level in the form of chromosome breaks and translocations, and at the molecular level predominately by multiple, large DNA deletions (4Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar).The gene responsible for the WS phenotype has been identified, cloned, and sequenced (6Yu C.-E. Oshima J. Fu Y.-H. Wijsman E.M. Hisama F. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1479) Google Scholar). The cDNA encodes a protein (hereafter designated as WRN) of 1432 amino acids with a central helicase domain that is homologous in sequence to members of the RecQ family of DNA helicases. This family includes Escherichia coli RecQ,Saccharomyces cerevisiae Sgs-1p, Schizosaccharomyces pombe Rqh-1p, human RecQL, and the protein associated with Bloom Syndrome, BLM. All of these proteins share the seven sequence motifs common to helicases, including the characteristic motif II DExH box (7Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar). The prototype of this family, the bacterial RecQ protein, is an active helicase that unwinds DNA in the 3′ → 5′ direction (8Umezu K. Nakayama K. Nakayama H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5363-5367Crossref PubMed Scopus (226) Google Scholar). Likewise, as predicted from DNA sequence alignments, the yeast Sgs-1p and the human RecQL, WRN, and BLM proteins have recently been demonstrated to exhibit ATP-dependent, 3′ → 5′ DNA unwinding activities in vitro (9Lu J. Mullen J.R. Brill S.J. Kleff S. Romeo A.M. Sternglanz R. Nature. 1996; 383: 678-679Crossref PubMed Scopus (138) Google Scholar, 10Bennett R.J. Sharp J.A. Wang J.C. J. Biol. Chem. 1998; 273: 9644-9650Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Tada S. Yanagisawa J. Sonoyama T. Miyajima A. Seki M. Ui M. Enomoto T. Cell Struct. Funct. 1996; 21: 123-132Crossref PubMed Scopus (12) Google Scholar, 12Gray M.D. Shen J.-C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (516) Google Scholar, 13Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Crossref PubMed Scopus (327) Google Scholar).The RecQ family of DNA helicases is believed to participate in numerous DNA transactions such as replication, recombination, and repair.E. coli RecQ is believed to initiate homologous recombination and suppress illegitimate recombination and the generation of aberrant recombination products (14Hanada K. Ukita T. Kohno Y. Saito K. Kato J-I. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3860-3865Crossref PubMed Scopus (239) Google Scholar, 15Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (235) Google Scholar). Further, RecQ is proposed to play a role in the reassembly of replication forks disrupted by UV lesions (16Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Crossref PubMed Scopus (180) Google Scholar, 17Courcelle J. Hanawalt P.C. FASEB J. 1997; 11 (abstr.): 1368Google Scholar). The phenotypes of mutations in the eukaryotic homologs also suggest their involvement in these processes.S. cerevisiae SGS-1 mutants display increased frequency of chromosome missegregation and recombination within ribosomal DNA repeats to generate extrachromosomal rDNA circles (18Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (616) Google Scholar, 19Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 20Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar, 21Sinclair D. Guarente L. Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar). Genetic studies indicate that S. pombe Rqh-1 is required to suppress inappropriate recombination that is essential for reversible S-phase arrest (22Stewart E. Chapman C.R. Al-Khodairy F. Carr A.E. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (326) Google Scholar). Mutations in human BLM, responsible for Bloom Syndrome, are associated with a high frequency of sister chromatid exchange, sensitivity to several DNA damaging agents, and a defect in DNA replication (23Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 80: 655-666Abstract Full Text PDF Scopus (1204) Google Scholar). Finally, mutations in WRN result in large DNA deletions, possibly as a result of chromosomal rearrangements, a prolonged S-phase of DNA replication, and sensitivity to the genotoxic agent, 4-nitroquinoline-1-oxide (4Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar, 24Gebhart E. Bauer R. Raub U. Schinzel M. Ruprecht K.W. Jonas J.B. Hum. Genet. 1988; 80: 135-139Crossref PubMed Scopus (138) Google Scholar, 25Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell. Res. 1992; 202: 267-273Crossref PubMed Scopus (186) Google Scholar, 26Fujiwara Y. Higashikawa T. Tatsumi M. J. Cell. Physiol. 1977; 92: 365-374Crossref PubMed Scopus (130) Google Scholar, 27Ogburn C.E. Oshima J. Poot M. Chen R. Hunt K.E. Gollahon K.A. Rabinovitch P.S. Martin G.M. Hum. Genet. 1997; 101: 121-125Crossref PubMed Scopus (162) Google Scholar). Together, these observations suggest that RecQ DNA helicases, including WRN, are involved in maintaining genomic stability, possibly by preventing deleterious recombination events from occurring during active DNA metabolism.The BLM and WRN proteins are both human RecQ-like DNA helicases. Yet, mutations in the respective genes result in distinctly different phenotypes. Examination of the domain structure of the two proteins reveals that, outside the helicase domain, the two polypeptides share little, if any, homology (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar). These regions, N- and C-terminal to the helicase domain, may encode other activities and/or interact with accessory proteins, both of which could determine the type of DNA transaction that each helicase carries out. Hence, identification of properties unique to each helicase may shed light on our understanding of their biological function(s). Recently, a conserved nuclease domain was identified in WRN by protein sequence data base searches (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 29Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Boork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (214) Google Scholar, 30Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (180) Google Scholar). This domain is unique to WRN, spans amino acids 80–240 in the N terminus, and exhibits significant similarity to the 3′ → 5′ proofreading domain of E. coli DNA polymerase I, to RNaseD and to the nuclease domain of the human polymyositis/scleroderma nuclear autoantigen. Based on this homology, WRN was proposed to encode an exonuclease. In this report we present molecular genetic, biochemical, and immunochemical evidence to demonstrate that the WRN protein possesses an integral DNA exonuclease activity in addition to its intrinsic DNA helicase activity.DISCUSSIONThe recent identification in WRN of a nuclease domain with a sequence consensus similar to the 3′ → 5′ proofreading domain of E. coli polymerase I and to RNase D (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 29Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Boork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (214) Google Scholar, 30Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (180) Google Scholar) led us to test whether WRN exhibits a deoxyribonucleolytic activity in vitro. Three complementary approaches independently indicate thatWRN encodes an endogenous exonucleolytic activity in addition to a DNA helicase activity.First we demonstrate a direct correlation between the presence of the putative WRN nuclease domain and observation of nuclease activity (Fig.1). Full-length wild-type WRN purified by a single step of His-Bind affinity chromatography exhibits both DNA helicase and DNA exonuclease activities. Mutant proteins harboring either a deletion of the helicase domain (WRN-ΔH) or a single-base substitution (WRN-K577M) that inactivates ATP hydrolysis digest DNA exonucleolytically but fail to unwind it. In contrast, WRN-ΔE lacking the nuclease domain is unable to degrade DNA but retains its ability to displace DNA. Evidence for the presence of an exonuclease has also been obtained by Huang et al. (34Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (374) Google Scholar). These authors site- specifically altered two critical amino acid residues (Asp-82 and Glu-84) in the N-terminal exonuclease domain that are predicted to be important for nuclease activity. While the nuclease activity was abolished in these mutant proteins, the helicase activity remained unchanged relative to wild-type WRN. These genetic studies provide convincing evidence that hydrolysis of DNA is dependent on an intact N-terminal WRN exonuclease domain and that it does not require an intact helicase domain.Second, our data provide biochemical evidence to suggest that the exonuclease and helicase activities reside on the WRN polypeptide. Both activities copurify with an extensively purified preparation of WRN. The elution profile of the exonuclease activity from the final step of His-Bind affinity chromatography is superimposable on the elution profiles of the 160-kDa WRN protein, the WRN DNA helicase and ATPase (Fig. 3). Furthermore, the WRN helicase and exonuclease activities co-migrate on glycerol gradients suggesting that both activities are encoded by a protein of a high molecular mass (Fig. 4). While most exonucleases are of low molecular weight, high molecular weight DNA polymerases frequently contain an associated 3′ → 5′ exonuclease that functions in proofreading errors by the polymerase (35Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). That the high molecular weight nuclease is not the result of such contaminating DNA polymerases was demonstrated by primer extension assays. No strand extension was observed when the DNA substrate used for helicase/exonuclease activity assays was incubated with WRN in the presence of all four dNTPs under DNA synthesis reaction conditions (not shown).Finally, by incubating WRN with a polyclonal anti-WRN antiserum, we demonstrate that the helicase and nuclease activities are co-immunoprecipitated in a complex with protein A-agarose (Fig. 5). The depletion of the enzymatic activities of WRN is specific since neither activity is precipitated by the preimmune serum obtained from the same animal. These results further suggest that the helicase and exonuclease activities reside on the WRN polypeptide.The presence of both a DNA helicase and a DNA exonuclease activity on the same polypeptide is so far unique to the WRN protein and may provide clues to its function in DNA metabolism. The fact that they are encoded within separate domains and that they can be uncoupled by selective deletion of each domain suggests that these activities need not function concertedly during the same catalytic step. Furthermore, studies with a mutant WRN protein in which the lysine residue (Lys-577) essential for NTP hydrolysis is substituted by a methionine residue show that the mutant protein can lose its ability to unwind DNA but retain its nuclease function. The spatial separation of helicase and exonuclease is reminiscent of many DNA polymerases in which polymerization and exonucleolytic hydrolysis occur sequentially (35Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). This could imply that the exonuclease functions in the removal of damaged DNA to yield a functional 3′-terminus suitable for elongation by a DNA polymerase that lacks proofreading exonucleolytic activity. Such a DNA polymerase presumably functions in concert with a 3′ → 5′ helicase that displaces duplex DNA ahead of the growing replication fork.The ability of WRN helicase to exonucleolytically digest DNA also distinguishes it from other members of the RecQ helicase family. Although all members share the conserved helicase domains (7Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar), none except WRN encodes an exonuclease domain. The presence of three, seemingly functionally redundant RecQ helicases in human cells (RecQL, BLM, and WRN) is puzzling. However, as more information is gained about possible other activities exhibited by these proteins, their substrate preferences, and their interaction with other proteins, they will likely be more distinct from one another. Hence, the helicase activity of WRN in conjunction with its exonuclease may limit the participation of WRN to processes that are distinct from those that require the RecQL and BLM proteins. Defective WRN activities could, therefore, impair these specific processes and result in the phenotypic manifestations that are characteristic of Werner Syndrome.In summary, we have identified a novel exonuclease that is encoded within the N-terminal sequence of the WRN protein. In the following paper (36Kamath-Loeb A.S. Shen J.-C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), we have biochemically characterized this activity and present evidence that it is a 3′ → 5′ exonuclease with properties distinct from other known exonucleases. The exonucleolytic activity exhibited by WRN could contribute to the unique characteristics of Werner Syndrome and distinguish it from other genomic instability disorders. Werner Syndrome (WS)1 is an inherited disease characterized by an early onset of atherosclerosis, osteoporosis, diabetes mellitus, and cancers of non-epithelial cell origin (1Epstein C.J. Martin G.M. Schultz A.L. Motulsky A.G. Medicine. 1966; 45: 177-221Crossref PubMed Scopus (735) Google Scholar, 2Goto M. Miller R.W. Ishikawa Y. Sugano H. Cancer Epidemiol. Biomark. Prev. 1996; 5: 239-246PubMed Google Scholar). Cultured cells from WS patients also present a shortened replicative lifespan (3Martin G.M. Sprague C.A. Epstein C.J. Lab. Invest. 1970; 23: 86-92PubMed Google Scholar) and increased genetic instability (4Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar). The genomic instability of WS cells is manifested at the cytogenetic level in the form of chromosome breaks and translocations, and at the molecular level predominately by multiple, large DNA deletions (4Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar). The gene responsible for the WS phenotype has been identified, cloned, and sequenced (6Yu C.-E. Oshima J. Fu Y.-H. Wijsman E.M. Hisama F. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1479) Google Scholar). The cDNA encodes a protein (hereafter designated as WRN) of 1432 amino acids with a central helicase domain that is homologous in sequence to members of the RecQ family of DNA helicases. This family includes Escherichia coli RecQ,Saccharomyces cerevisiae Sgs-1p, Schizosaccharomyces pombe Rqh-1p, human RecQL, and the protein associated with Bloom Syndrome, BLM. All of these proteins share the seven sequence motifs common to helicases, including the characteristic motif II DExH box (7Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar). The prototype of this family, the bacterial RecQ protein, is an active helicase that unwinds DNA in the 3′ → 5′ direction (8Umezu K. Nakayama K. Nakayama H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5363-5367Crossref PubMed Scopus (226) Google Scholar). Likewise, as predicted from DNA sequence alignments, the yeast Sgs-1p and the human RecQL, WRN, and BLM proteins have recently been demonstrated to exhibit ATP-dependent, 3′ → 5′ DNA unwinding activities in vitro (9Lu J. Mullen J.R. Brill S.J. Kleff S. Romeo A.M. Sternglanz R. Nature. 1996; 383: 678-679Crossref PubMed Scopus (138) Google Scholar, 10Bennett R.J. Sharp J.A. Wang J.C. J. Biol. Chem. 1998; 273: 9644-9650Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 11Tada S. Yanagisawa J. Sonoyama T. Miyajima A. Seki M. Ui M. Enomoto T. Cell Struct. Funct. 1996; 21: 123-132Crossref PubMed Scopus (12) Google Scholar, 12Gray M.D. Shen J.-C. Kamath-Loeb A.S. Blank A. Sopher B.L. Martin G.M. Oshima J. Loeb L.A. Nat. Genet. 1997; 17: 100-103Crossref PubMed Scopus (516) Google Scholar, 13Karow J.K. Chakraverty R.K. Hickson I.D. J. Biol. Chem. 1997; 272: 30611-30614Crossref PubMed Scopus (327) Google Scholar). The RecQ family of DNA helicases is believed to participate in numerous DNA transactions such as replication, recombination, and repair.E. coli RecQ is believed to initiate homologous recombination and suppress illegitimate recombination and the generation of aberrant recombination products (14Hanada K. Ukita T. Kohno Y. Saito K. Kato J-I. Ikeda H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3860-3865Crossref PubMed Scopus (239) Google Scholar, 15Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Crossref PubMed Scopus (235) Google Scholar). Further, RecQ is proposed to play a role in the reassembly of replication forks disrupted by UV lesions (16Courcelle J. Carswell-Crumpton C. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3714-3719Crossref PubMed Scopus (180) Google Scholar, 17Courcelle J. Hanawalt P.C. FASEB J. 1997; 11 (abstr.): 1368Google Scholar). The phenotypes of mutations in the eukaryotic homologs also suggest their involvement in these processes.S. cerevisiae SGS-1 mutants display increased frequency of chromosome missegregation and recombination within ribosomal DNA repeats to generate extrachromosomal rDNA circles (18Gangloff S. McDonald J.P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (616) Google Scholar, 19Watt P.M. Louis E.J. Borts R.H. Hickson I.D. Cell. 1995; 81: 253-260Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 20Watt P.M. Hickson I.D. Borts R.H. Louis E.J. Genetics. 1996; 144: 935-945Crossref PubMed Google Scholar, 21Sinclair D. Guarente L. Cell. 1997; 91: 1033-1042Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar). Genetic studies indicate that S. pombe Rqh-1 is required to suppress inappropriate recombination that is essential for reversible S-phase arrest (22Stewart E. Chapman C.R. Al-Khodairy F. Carr A.E. Enoch T. EMBO J. 1997; 16: 2682-2692Crossref PubMed Scopus (326) Google Scholar). Mutations in human BLM, responsible for Bloom Syndrome, are associated with a high frequency of sister chromatid exchange, sensitivity to several DNA damaging agents, and a defect in DNA replication (23Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 80: 655-666Abstract Full Text PDF Scopus (1204) Google Scholar). Finally, mutations in WRN result in large DNA deletions, possibly as a result of chromosomal rearrangements, a prolonged S-phase of DNA replication, and sensitivity to the genotoxic agent, 4-nitroquinoline-1-oxide (4Salk D. Au K. Hoehn H. Martin G.M. Cytogenet. Cell Genet. 1981; 30: 92-107Crossref PubMed Scopus (210) Google Scholar, 5Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar, 24Gebhart E. Bauer R. Raub U. Schinzel M. Ruprecht K.W. Jonas J.B. Hum. Genet. 1988; 80: 135-139Crossref PubMed Scopus (138) Google Scholar, 25Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell. Res. 1992; 202: 267-273Crossref PubMed Scopus (186) Google Scholar, 26Fujiwara Y. Higashikawa T. Tatsumi M. J. Cell. Physiol. 1977; 92: 365-374Crossref PubMed Scopus (130) Google Scholar, 27Ogburn C.E. Oshima J. Poot M. Chen R. Hunt K.E. Gollahon K.A. Rabinovitch P.S. Martin G.M. Hum. Genet. 1997; 101: 121-125Crossref PubMed Scopus (162) Google Scholar). Together, these observations suggest that RecQ DNA helicases, including WRN, are involved in maintaining genomic stability, possibly by preventing deleterious recombination events from occurring during active DNA metabolism. The BLM and WRN proteins are both human RecQ-like DNA helicases. Yet, mutations in the respective genes result in distinctly different phenotypes. Examination of the domain structure of the two proteins reveals that, outside the helicase domain, the two polypeptides share little, if any, homology (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar). These regions, N- and C-terminal to the helicase domain, may encode other activities and/or interact with accessory proteins, both of which could determine the type of DNA transaction that each helicase carries out. Hence, identification of properties unique to each helicase may shed light on our understanding of their biological function(s). Recently, a conserved nuclease domain was identified in WRN by protein sequence data base searches (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 29Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Boork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (214) Google Scholar, 30Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (180) Google Scholar). This domain is unique to WRN, spans amino acids 80–240 in the N terminus, and exhibits significant similarity to the 3′ → 5′ proofreading domain of E. coli DNA polymerase I, to RNaseD and to the nuclease domain of the human polymyositis/scleroderma nuclear autoantigen. Based on this homology, WRN was proposed to encode an exonuclease. In this report we present molecular genetic, biochemical, and immunochemical evidence to demonstrate that the WRN protein possesses an integral DNA exonuclease activity in addition to its intrinsic DNA helicase activity. DISCUSSIONThe recent identification in WRN of a nuclease domain with a sequence consensus similar to the 3′ → 5′ proofreading domain of E. coli polymerase I and to RNase D (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 29Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Boork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (214) Google Scholar, 30Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (180) Google Scholar) led us to test whether WRN exhibits a deoxyribonucleolytic activity in vitro. Three complementary approaches independently indicate thatWRN encodes an endogenous exonucleolytic activity in addition to a DNA helicase activity.First we demonstrate a direct correlation between the presence of the putative WRN nuclease domain and observation of nuclease activity (Fig.1). Full-length wild-type WRN purified by a single step of His-Bind affinity chromatography exhibits both DNA helicase and DNA exonuclease activities. Mutant proteins harboring either a deletion of the helicase domain (WRN-ΔH) or a single-base substitution (WRN-K577M) that inactivates ATP hydrolysis digest DNA exonucleolytically but fail to unwind it. In contrast, WRN-ΔE lacking the nuclease domain is unable to degrade DNA but retains its ability to displace DNA. Evidence for the presence of an exonuclease has also been obtained by Huang et al. (34Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (374) Google Scholar). These authors site- specifically altered two critical amino acid residues (Asp-82 and Glu-84) in the N-terminal exonuclease domain that are predicted to be important for nuclease activity. While the nuclease activity was abolished in these mutant proteins, the helicase activity remained unchanged relative to wild-type WRN. These genetic studies provide convincing evidence that hydrolysis of DNA is dependent on an intact N-terminal WRN exonuclease domain and that it does not require an intact helicase domain.Second, our data provide biochemical evidence to suggest that the exonuclease and helicase activities reside on the WRN polypeptide. Both activities copurify with an extensively purified preparation of WRN. The elution profile of the exonuclease activity from the final step of His-Bind affinity chromatography is superimposable on the elution profiles of the 160-kDa WRN protein, the WRN DNA helicase and ATPase (Fig. 3). Furthermore, the WRN helicase and exonuclease activities co-migrate on glycerol gradients suggesting that both activities are encoded by a protein of a high molecular mass (Fig. 4). While most exonucleases are of low molecular weight, high molecular weight DNA polymerases frequently contain an associated 3′ → 5′ exonuclease that functions in proofreading errors by the polymerase (35Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). That the high molecular weight nuclease is not the result of such contaminating DNA polymerases was demonstrated by primer extension assays. No strand extension was observed when the DNA substrate used for helicase/exonuclease activity assays was incubated with WRN in the presence of all four dNTPs under DNA synthesis reaction conditions (not shown).Finally, by incubating WRN with a polyclonal anti-WRN antiserum, we demonstrate that the helicase and nuclease activities are co-immunoprecipitated in a complex with protein A-agarose (Fig. 5). The depletion of the enzymatic activities of WRN is specific since neither activity is precipitated by the preimmune serum obtained from the same animal. These results further suggest that the helicase and exonuclease activities reside on the WRN polypeptide.The presence of both a DNA helicase and a DNA exonuclease activity on the same polypeptide is so far unique to the WRN protein and may provide clues to its function in DNA metabolism. The fact that they are encoded within separate domains and that they can be uncoupled by selective deletion of each domain suggests that these activities need not function concertedly during the same catalytic step. Furthermore, studies with a mutant WRN protein in which the lysine residue (Lys-577) essential for NTP hydrolysis is substituted by a methionine residue show that the mutant protein can lose its ability to unwind DNA but retain its nuclease function. The spatial separation of helicase and exonuclease is reminiscent of many DNA polymerases in which polymerization and exonucleolytic hydrolysis occur sequentially (35Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). This could imply that the exonuclease functions in the removal of damaged DNA to yield a functional 3′-terminus suitable for elongation by a DNA polymerase that lacks proofreading exonucleolytic activity. Such a DNA polymerase presumably functions in concert with a 3′ → 5′ helicase that displaces duplex DNA ahead of the growing replication fork.The ability of WRN helicase to exonucleolytically digest DNA also distinguishes it from other members of the RecQ helicase family. Although all members share the conserved helicase domains (7Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar), none except WRN encodes an exonuclease domain. The presence of three, seemingly functionally redundant RecQ helicases in human cells (RecQL, BLM, and WRN) is puzzling. However, as more information is gained about possible other activities exhibited by these proteins, their substrate preferences, and their interaction with other proteins, they will likely be more distinct from one another. Hence, the helicase activity of WRN in conjunction with its exonuclease may limit the participation of WRN to processes that are distinct from those that require the RecQL and BLM proteins. Defective WRN activities could, therefore, impair these specific processes and result in the phenotypic manifestations that are characteristic of Werner Syndrome.In summary, we have identified a novel exonuclease that is encoded within the N-terminal sequence of the WRN protein. In the following paper (36Kamath-Loeb A.S. Shen J.-C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), we have biochemically characterized this activity and present evidence that it is a 3′ → 5′ exonuclease with properties distinct from other known exonucleases. The exonucleolytic activity exhibited by WRN could contribute to the unique characteristics of Werner Syndrome and distinguish it from other genomic instability disorders. The recent identification in WRN of a nuclease domain with a sequence consensus similar to the 3′ → 5′ proofreading domain of E. coli polymerase I and to RNase D (28Morozov V. Mushegian A.R. Koonin E.V. Boork P. Trends Biochem. Sci. 1997; 22: 417-418Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 29Mushegian A.R. Bassett Jr., D.E. Boguski M.S. Boork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (214) Google Scholar, 30Mian I.S. Nucleic Acids Res. 1997; 25: 3187-3195Crossref PubMed Scopus (180) Google Scholar) led us to test whether WRN exhibits a deoxyribonucleolytic activity in vitro. Three complementary approaches independently indicate thatWRN encodes an endogenous exonucleolytic activity in addition to a DNA helicase activity. First we demonstrate a direct correlation between the presence of the putative WRN nuclease domain and observation of nuclease activity (Fig.1). Full-length wild-type WRN purified by a single step of His-Bind affinity chromatography exhibits both DNA helicase and DNA exonuclease activities. Mutant proteins harboring either a deletion of the helicase domain (WRN-ΔH) or a single-base substitution (WRN-K577M) that inactivates ATP hydrolysis digest DNA exonucleolytically but fail to unwind it. In contrast, WRN-ΔE lacking the nuclease domain is unable to degrade DNA but retains its ability to displace DNA. Evidence for the presence of an exonuclease has also been obtained by Huang et al. (34Huang S. Li B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (374) Google Scholar). These authors site- specifically altered two critical amino acid residues (Asp-82 and Glu-84) in the N-terminal exonuclease domain that are predicted to be important for nuclease activity. While the nuclease activity was abolished in these mutant proteins, the helicase activity remained unchanged relative to wild-type WRN. These genetic studies provide convincing evidence that hydrolysis of DNA is dependent on an intact N-terminal WRN exonuclease domain and that it does not require an intact helicase domain. Second, our data provide biochemical evidence to suggest that the exonuclease and helicase activities reside on the WRN polypeptide. Both activities copurify with an extensively purified preparation of WRN. The elution profile of the exonuclease activity from the final step of His-Bind affinity chromatography is superimposable on the elution profiles of the 160-kDa WRN protein, the WRN DNA helicase and ATPase (Fig. 3). Furthermore, the WRN helicase and exonuclease activities co-migrate on glycerol gradients suggesting that both activities are encoded by a protein of a high molecular mass (Fig. 4). While most exonucleases are of low molecular weight, high molecular weight DNA polymerases frequently contain an associated 3′ → 5′ exonuclease that functions in proofreading errors by the polymerase (35Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). That the high molecular weight nuclease is not the result of such contaminating DNA polymerases was demonstrated by primer extension assays. No strand extension was observed when the DNA substrate used for helicase/exonuclease activity assays was incubated with WRN in the presence of all four dNTPs under DNA synthesis reaction conditions (not shown). Finally, by incubating WRN with a polyclonal anti-WRN antiserum, we demonstrate that the helicase and nuclease activities are co-immunoprecipitated in a complex with protein A-agarose (Fig. 5). The depletion of the enzymatic activities of WRN is specific since neither activity is precipitated by the preimmune serum obtained from the same animal. These results further suggest that the helicase and exonuclease activities reside on the WRN polypeptide. The presence of both a DNA helicase and a DNA exonuclease activity on the same polypeptide is so far unique to the WRN protein and may provide clues to its function in DNA metabolism. The fact that they are encoded within separate domains and that they can be uncoupled by selective deletion of each domain suggests that these activities need not function concertedly during the same catalytic step. Furthermore, studies with a mutant WRN protein in which the lysine residue (Lys-577) essential for NTP hydrolysis is substituted by a methionine residue show that the mutant protein can lose its ability to unwind DNA but retain its nuclease function. The spatial separation of helicase and exonuclease is reminiscent of many DNA polymerases in which polymerization and exonucleolytic hydrolysis occur sequentially (35Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Company, New York1992Google Scholar). This could imply that the exonuclease functions in the removal of damaged DNA to yield a functional 3′-terminus suitable for elongation by a DNA polymerase that lacks proofreading exonucleolytic activity. Such a DNA polymerase presumably functions in concert with a 3′ → 5′ helicase that displaces duplex DNA ahead of the growing replication fork. The ability of WRN helicase to exonucleolytically digest DNA also distinguishes it from other members of the RecQ helicase family. Although all members share the conserved helicase domains (7Ellis N.A. Curr. Opin. Genet. Dev. 1997; 7: 354-363Crossref PubMed Scopus (115) Google Scholar), none except WRN encodes an exonuclease domain. The presence of three, seemingly functionally redundant RecQ helicases in human cells (RecQL, BLM, and WRN) is puzzling. However, as more information is gained about possible other activities exhibited by these proteins, their substrate preferences, and their interaction with other proteins, they will likely be more distinct from one another. Hence, the helicase activity of WRN in conjunction with its exonuclease may limit the participation of WRN to processes that are distinct from those that require the RecQL and BLM proteins. Defective WRN activities could, therefore, impair these specific processes and result in the phenotypic manifestations that are characteristic of Werner Syndrome. In summary, we have identified a novel exonuclease that is encoded within the N-terminal sequence of the WRN protein. In the following paper (36Kamath-Loeb A.S. Shen J.-C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), we have biochemically characterized this activity and present evidence that it is a 3′ → 5′ exonuclease with properties distinct from other known exonucleases. The exonucleolytic activity exhibited by WRN could contribute to the unique characteristics of Werner Syndrome and distinguish it from other genomic instability disorders." @default.
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