Baomin Li

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging. The gene responsible for the syndrome was recently cloned and shown to encode a protein with strong homology to DNA/RNA helicases. In addition, the Werner syndrome protein (WRN) possesses an exonuclease activity. Based on the homology to helicases it has been proposed that WRN functions in some aspects of DNA replication, recombination, or repair. However, there is currently no evidence of a role of WRN in any of these processes; therefore, its biological function remains unknown. Using a biochemical approach, we have identified two polypeptides that bind to the WRN protein. Peptide sequence analysis indicates that the two proteins are identical to Ku70 and Ku80, a heterodimer involved in double strand DNA break repair by non-homologous DNA end joining. Protein-protein interaction studies reveal that WRN binds directly to Ku80 and that this interaction is mediated by the amino terminus of WRN. In addition, we show that the binding of Ku alters the specificity of the WRN exonuclease. These results suggest a potential involvement of WRN in the repair of double strand DNA breaks.

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN(H), a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRN-associated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD(+), PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.

BACKGROUND AND PURPOSE: Although recent trials have suggested that stenting is worse than medical therapy for patients with severe symptomatic intracranial atherosclerotic stenosis, it is not clear whether this conclusion applies to a subset of patients with hypoperfusion symptoms. To justify for a new trial in China, we performed a multicenter prospective registry study to evaluate the safety and efficacy of endovascular stenting within 30 days for patients with severe symptomatic intracranial atherosclerotic stenosis. METHODS: Patients with symptomatic intracranial atherosclerotic stenosis caused by 70% to 99% stenosis combined with poor collaterals were enrolled. The patients were treated either with balloon-mounted stent or with balloon predilation plus self-expanding stent as determined by the operators following a guideline. The primary outcome within 30 days is stroke, transient ischemic attack, and death after stenting. The secondary outcome is successful revascularization. The baseline characteristics and outcomes of the 2 treatment groups were compared. RESULTS: From September 2013 to January 2015, among 354 consecutive patients, 300 patients (aged 58.3±9.78 years) were recruited, including 159 patients treated with balloon-mounted stent and 141 patients with balloon plus self-expanding stent. The 30-day rate of stroke, transient ischemic attack, and death was 4.3%. Successful revascularization was 97.3%. Patients treated with balloon-mounted stent were older, less likely to have middle cerebral artery lesions, more likely to have vertebral artery lesions, more likely to have Mori A lesions, less likely to have Mori C lesions, and likely to have lower degree of residual stenosis than patients treated with balloon plus self-expanding stent. CONCLUSIONS: The short-term safety and efficacy of endovascular stenting for patients with severe symptomatic intracranial atherosclerotic stenosis in China is acceptable. Balloon-mounted stent may have lower degree of residual stenosis than self-expanding stent. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01968122.

Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends. Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends. Werner syndrome Werner syndrome protein double strand DNA breaks glutathione S-transferase polyacrylamide gel electrophoresis Werner syndrome (WS)1 is a rare disorder characterized by the early appearance of many diseases characteristics of human aging such as atherosclerosis, osteoporosis and diabetes mellitus type II (1Epstein C.J. Martin G.M. Schultz A.L. Motulsky A.G. Medicine. 1966; 45: 177-221Crossref PubMed Scopus (745) Google Scholar, 2Dyer C. Sinclair A. Age Ageing. 1998; 27: 73-80Crossref PubMed Scopus (38) Google Scholar, 3Martin G.M. Birth Defects Orig. Artic. Ser. 1978; 14: 5-39PubMed Google Scholar). In addition, WS patients are prone to many types of soft tissue tumors (4Goto M. Miller R.W. Ishikawa Y. Sugano H. Cancer Epidemiol. Biomark. Prev. 1996; 5: 239-246PubMed Google Scholar). Cells from WS patients display a shortened replicative life span and elevated levels of chromosomal abnormalities, including insertions, deletions and translocations (5Fukuchi K. Martin G.M. Monnat R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (391) Google Scholar, 6Salk D. Au K. Hoehn H. Martin G.M. Adv. Exp. Med. Biol. 1985; 190: 541-550Crossref PubMed Scopus (53) Google Scholar). The gene responsible for WS has been cloned and encodes a protein of 1,432 amino acids (WRN) (7Yu C.E. Oshima J. Fu Y.H. Wijsman E. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G. Mulligan J. Schellenberg G. Science. 1996; 272: 258-262Crossref PubMed Scopus (1496) Google Scholar). A nuclear localization signal has been identified at its carboxyl-terminal end (8Matsumoto T. Shimamoto A. Goto M. Furuichi Y. Nat. Genet. 1997; 16: 335-336Crossref PubMed Scopus (161) Google Scholar), and all the described WS mutations result in a predicted truncated protein that fails to enter the nucleus. The central region of WRN is homologous to a seven-motif domain found in helicases from a wide variety of organisms, including bacteria (recQ), yeast (Sgs1), and human (Bloom syndrome (BLM), RecQL) (7Yu C.E. Oshima J. Fu Y.H. Wijsman E. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G. Mulligan J. Schellenberg G. Science. 1996; 272: 258-262Crossref PubMed Scopus (1496) Google Scholar, 9Ellis N.A. Groden T.Z. Ye J. Straughen D.J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1221) Google Scholar, 10Puranam K.L. Blackshear P.J. J. Biol. Chem. 1994; 269: 29838-29845Abstract Full Text PDF PubMed Google Scholar). On the other hand, the amino-terminal region of WRN is quite unique among this family of helicases because it contains a sequence that resembles the exonuclease domain of Escherichia coli RNA polymerase I and RNase D (11Mushegian A.R. Bassett D.E. Boguski M.S. Bork P. Koonin E.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5831-5836Crossref PubMed Scopus (216) Google Scholar,12Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (204) Google Scholar). Indeed, studies with the purified recombinant protein have shown that WRN displays both 3′- to 5′-exonuclease and 3′- to 5′-helicase activities (13Huang S. Li B. Gray M. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar, 14Shen J.C. Gray M. Oshima J. Loeb L. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar, 15Shen J.C. Gray M. Oshima J. Kamath-Loeb A. Fry M. Loeb L. J. Biol. Chem. 1998; 273: 34139-34144Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). We have shown recently that Ku interacts with and alters the specificity of the WRN exonuclease (16Li B. Comai L. J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). These results indicated that in the presence of Ku, WRN degradation of 3′-recessed strand of a partial DNA duplex is strongly stimulated. In addition, Ku alters the specificity of WRN so that the blunt end DNA duplex and 3′-protruding DNA are also hydrolyzed by WRN exonuclease. Ku is a 70- and 80-kDa heterodimer that binds to the ends of double strand DNA and translocates along the DNA in an ATP-independent manner, allowing several Ku heterodimers to bind to a single DNA molecule (17Devries E.W. Vandriel W.C. Bergsma A.C. Vandervliet P.C. J. Mol. Biol. 1989; 208: 65-78Crossref PubMed Scopus (217) Google Scholar, 18Mimori T. Hardin J.A. J. Biol. Chem. 1986; 261: 375-379Google Scholar, 19Ochem A.E. Skopac D. Costa M. Rabilloud T. Vuillard L. Simoncsits A. Giacca M. Falaschi A. J. Biol. Chem. 1997; 272: 29919-29926Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Pailard S. Strauss F. Nucleic Acids Res. 1991; 19: 5619-5624Crossref PubMed Scopus (188) Google Scholar). In higher eukaryotes, Ku has been implicated in the metabolism of DNA ends, including the repair of double strand DNA breaks (DSBs) and the V(D)J recombination process (21Karran P. Curr. Opin. Genet. Dev. 2000; 10: 144-150Crossref PubMed Scopus (388) Google Scholar, 22Kanaar R. Hoeijmakers J. van Gent D.C. Cell Biol. 1998; 8: 483-491Scopus (458) Google Scholar). In mammalian cells, Ku appears to be the first protein to recognize DSBs. Upon binding to the ends of broken DNA, Ku interacts with the DNA-dependent protein kinase catalytic subunit, stabilizes the interaction of this catalytic subunit with DNA, and enhances its protein kinase activity (18Mimori T. Hardin J.A. J. Biol. Chem. 1986; 261: 375-379Google Scholar, 21Karran P. Curr. Opin. Genet. Dev. 2000; 10: 144-150Crossref PubMed Scopus (388) Google Scholar, 23Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar, 24Hammarsten O. Chu G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 525-530Crossref PubMed Scopus (202) Google Scholar). The subsequent steps in the end-joining process are then thought to require the recruitment of specific activities necessary for the processing and ligation of DNA ends (25Baumann P. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14066-14070Crossref PubMed Scopus (273) Google Scholar, 26Bailey S.M. Meyne J. Chen D., J. Kurimasa A. Li G.C. Lehnert B.E. Goodwin E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14899-14904Crossref PubMed Scopus (349) Google Scholar, 27Critchlow S. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 28Ramsden D.A. Gellert M. EMBO J. 1998; 17: 609-614Crossref PubMed Scopus (248) Google Scholar). Although factors such as XRCC4 and DNA ligase IV have been shown to be required for the ligation step, the processing of DNA ends is still poorly understood, and the identity of the nucleases involved in this process remains to be defined. Interestingly, the finding that Ku interacts with and stimulates the activity of the WRN exonuclease suggests that WRN may be one of the factors that function in the processing of broken DNA ends. In this study, to understand better the functional relevance of this finding, we have characterized the interaction of WRN with Ku and DNA ends. First, we show that Ku interacts with the extreme amino terminus of WRN, from amino acid 1 to 50, whereas the central region of Ku80, from amino acid 215 to 276, is necessary for binding to WRN. Then, we demonstrate that Ku can efficiently stimulate the exonuclease activity of an amino-terminal mutant WRN (WRNΔC388; amino acids 1–388), indicating that the helicase and carboxyl-terminal domains of WRN are dispensable for Ku-stimulated processing of DNA ends. Moreover, using electrophoretic mobility shift assays we show that WRN forms a strong complex with Ku on DNA. Recombinant and a of deletion mutants purified as described in B. Comai L. J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google recombinant WRN to in purified a and by on and Ku80 in cells, and the Ku70/80 heterodimer purified by and DNA The the WRN with a at amino acid using a polymerase The with amino acids and by polymerase using the DNA and The with and a at then cloned WRN the the of Recombinant DNA purified and with DNA to the recombinant Ku80 of of Ku80 to be in the of the RNA polymerase These then to in in the presence of using a of on and then with of in Ku80 mutants and of and by in and on gel by by of the and as in E. coli and amino acid the of purified protein and on the and then with of from Ku80 of Ku80 in the at from the using 1 1 a of and by and with In a of WRN protein and on and then with of from with and the from the using and then by and DNA exonuclease activity as described in (16Li B. Comai L. J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google to and to at the with and The by by to 1 of DNA and of Ku80, WRN, and in a of The at for and then by the of of a at for the DNA by gel electrophoresis and by using and kinase and then to a DNA with of Ku and WRN in of and at for Then, the by electrophoresis a polyacrylamide gel at in the The on and to gel assays, of Ku and of WRN first with DNA at for the described Then, of Ku80 WRN to the a at the on a polyacrylamide gel and as described We have shown that the amino-terminal region of WRN interacts with Ku80 (16Li B. Comai L. J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). the amino acid domain of WRN necessary for this we assays using of the amino-terminal region of WRN and 1 In addition, in the as protein and on glutathione and in assays with from with a recombinant the on a and by with Ku80 with WRN In addition, Ku80 by the protein the region of WRN from amino acid 1 to 50 whereas WRN amino acids amino acids a protein to interact with These results indicate that the region of WRN between amino acids 1 and 50 is necessary and sufficient for binding to demonstrate further that this region of WRN with an Ku70/80 of the and with with and the with and The results of this that the amino-terminal domain acids is sufficient for binding to the Ku70/80 heterodimer 1 results purified in to this and the region of Ku80 necessary for the interaction with WRN, various deletion mutants of Ku80 in in the presence of The in then with WRN a polymerase that on on a and by that deletion of the carboxyl-terminal region of Ku80, from amino acid to not the binding to WRN further deletion to amino acid the interaction of the amino terminus of Ku80, from amino acids 1 to also the interaction with WRN The polymerase not interact with of the Ku80 mutants in this not these results indicate that the central region of Ku80, from amino acids 215 to 276, is important for the interaction with region of Ku80 between amino acids 215 and 276 is necessary for the interaction with WRN. Ku80 and in the protein binding gel that of protein in binding WRN on that then with Ku80 mutants as described The by and by WRN exonuclease activity its amino terminus (13Huang S. Li B. Gray M. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar, A. J.C. Loeb L. Fry M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), whereas the helicase domain is in the central of the protein (7Yu C.E. Oshima J. Fu Y.H. Wijsman E. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G. Mulligan J. Schellenberg G. Science. 1996; 272: 258-262Crossref PubMed Scopus (1496) Google Scholar, 14Shen J.C. Gray M. Oshima J. Loeb L. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar, 15Shen J.C. Gray M. Oshima J. Kamath-Loeb A. Fry M. Loeb L. J. Biol. Chem. 1998; 273: 34139-34144Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). A also found that the amino-terminal domain of WRN, from amino acids 1 to is sufficient for the degradation of DNA with a (13Huang S. Li B. Gray M. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar). our results indicate that this region of WRN also binds to Ku80 we in a mutant of WRN the first amino acids and the helicase domain, is sufficient for of DNA mutant WRN using a and then in exonuclease assays with several DNA in the presence of Ku80, Ku70/80. hydrolyzed the 3′-recessed strand of a partial DNA duplex and the of blunt end DNA and in the presence of the exonuclease activity of strongly stimulated The in exonuclease activity on the presence of Ku70/80 because Ku80 to stimulate and in the presence of the Ku70/80 efficiently 3′-protruding ends and single strand DNA and that the DNA degradation is by WRN and not by a activity in the Ku we the exonuclease in the presence of a mutant of WRN exonuclease activity (13Huang S. Li B. Gray M. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google Scholar). shown in exonuclease activity in the presence of Ku80 Ku70/80 of and Ku80 that the DNA degradation on the presence of a functional WRN exonuclease. In Ku stimulates and alters the exonuclease activity of WRN the helicase domain, that DNA is not required in this process. WRN contains amino acid and helicase activities (7Yu C.E. Oshima J. Fu Y.H. Wijsman E. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G. Mulligan J. Schellenberg G. Science. 1996; 272: 258-262Crossref PubMed Scopus (1496) Google Scholar, 14Shen J.C. Gray M. Oshima J. Loeb L. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (182) Google Scholar). has also been shown that the helicase activity of WRN is on the of and stimulates the activity of the WRN exonuclease A. J.C. Loeb L. Fry M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.C. Kamath-Loeb A. A. B. Martin G. Oshima J. Loeb L. Nat. Genet. 1997; 17: PubMed Scopus Google Scholar). investigate further the of of the WRN exonuclease activity by Ku, we then is required for this stimulatory process. shown in in the presence of Ku, the of on the activity of WRN indicating that Ku-mediated of WRN exonuclease activity not require Ku has specificity for DNA substrates, it binds to the ends of double strand DNA, and has for and blunt end DNA (18Mimori T. Hardin J.A. J. Biol. Chem. 1986; 261: 375-379Google Scholar, S. EMBO J. 1997; 16: PubMed Scopus Google Scholar). On the other hand, WRN binds to double strand DNA A. Gray Nucleic Acids Res. 1999; 27: PubMed Scopus Google Scholar, Loeb Nucleic Acids Res. 2000; PubMed Scopus Google Scholar). we electrophoretic mobility shift to Ku the recruitment of WRN to DNA Upon of a DNA duplex with purified Ku, we the of 1 and one Ku70/80 heterodimers binding to one DNA In WRN to bind to DNA, as by the of complex on the gel and both WRN and Ku with the DNA in to the 1 and a in the gel complex as as the complex by Moreover, the of WRN the whereas the an protein on the mobility of of the these results indicate that can a complex on DNA in the presence of Ku, further the that Ku recruits WRN to DNA ends specific studies demonstrated that WRN an 3′- to 5′-exonuclease activity that 3′-recessed DNA (13Huang S. Li B. Gray M. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Crossref PubMed Scopus (376) Google J.C. Gray M. Oshima J. Kamath-Loeb A. Fry M. Loeb L. J. Biol. Chem. 1998; 273: 34139-34144Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). our has indicated that WRN interacts with Ku70/80 and Ku70/80 stimulates and alters the exonuclease activity of WRN, so that it can and blunt end DNA efficiently (16Li B. Comai L. J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). WRN not bind to double strand DNA and has a for single strand DNA A. Gray Nucleic Acids Res. 1999; 27: PubMed Scopus Google Scholar), our finding the that Ku may function as a factor that WRN in to its DNA Ku-mediated recruitment of WRN may a in the and subsequent activation of WRN exonuclease on specific DNA In this study, to understand better the functional between Ku and WRN, we have a of the between these Our that the interaction between WRN and Ku80 requires a of 50 amino acids at the extreme amino terminus of WRN. These results are in with the data by O. D. Dev. 2000; 14: Google that Ku interacts with the carboxyl-terminal region of WRN. our we have a interaction between the carboxyl-terminal domain of WRN and we have (16Li B. Comai L. J. Biol. Chem. 2000; 275: 28349-28352Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), because the terminus of WRN can with the protein O. D. Dev. 2000; 14: Google Scholar), it is that cellular WRN may have the interaction in Our of Ku80 mutants has also the of a region between amino acids 215 and 276 as an important domain required for Ku80 binding to WRN. region is from the of Ku80 which have been found to be involved in with and a DNA-dependent protein kinase catalytic subunit Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar, D. Jackson S.P. Nucleic Acids Res. 1999; 27: PubMed Scopus Google Scholar, Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). the region of WRN which binds to Ku80 is to the WRN exonuclease domain, we that the amino-terminal region of WRN be sufficient for Ku of the exonuclease activity. this we the of Ku on the exonuclease activity of a of WRN which is the helicase domain and the carboxyl-terminal Our results indicate that poorly 3′-recessed ends and blunt end DNA in the presence of Ku it efficiently ends and blunt end DNA as as single strand DNA. The activity is on the presence of a functional WRN because Ku70/80 not stimulate a mutant protein exonuclease activity. In addition, our results show that is dispensable for this stimulatory process. Ku-stimulated WRN exonuclease activity is and from the helicase domain and the terminus of WRN and not require investigate WRN can with its DNA we then gel shift These show that WRN by has DNA binding in the presence of Ku it forms a strong complex with DNA. These results that protein between WRN and Ku play a role in the recruitment of WRN to DNA ends and suggest a function in the processing of DSBs. In cells, by such as the of the and not can to (21Karran P. Curr. Opin. Genet. Dev. 2000; 10: 144-150Crossref PubMed Scopus (388) Google Scholar, 27Critchlow S. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). In the Ku DNA-dependent protein kinase catalytic subunit, and the complex are involved the repair of double strand DNA breaks by end T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar, 24Hammarsten O. Chu G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 525-530Crossref PubMed Scopus (202) Google Scholar, S. Nucleic Acids Res. 1998; 26: PubMed Scopus Google Scholar, U. M. P. M. 1997; PubMed Scopus Google Scholar). In addition, because ligation can be to the several studies have that activities are necessary in the repair process to the DNA the ends are U. M. Curr. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Gellert M. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). These nucleases recognize the broken DNA ends be to the of DNA by specific a 3′- to 5′-exonuclease and has been implicated in one DNA repair (21Karran P. Curr. Opin. Genet. Dev. 2000; 10: 144-150Crossref PubMed Scopus (388) Google Scholar, Gellert M. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and an protein to bind to DNA and a complex Gellert M. Dev. 1999; PubMed Scopus Google Scholar). complex displays several including partial of the DNA duplex and of the 3′-protruding end of double strand DNA. it has also been shown that can the of ends, that may DNA end at of Gellert M. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). a role in end remains WRN not bind to double strand it interacts with Ku and is to DNA ends by specific it is to that WRN, may in a of DNA repair which requires the processing of DNA ends are the it has that several of in mammalian (21Karran P. Curr. Opin. Genet. Dev. 2000; 10: 144-150Crossref PubMed Scopus (388) Google Scholar, U. M. Curr. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). it is that of these a of may also a of unique and are to are to other such as Y. T. M. J. Cell. PubMed Scopus Google Scholar, M. S. P. G. E. F. Res. 1989; PubMed Scopus Google Scholar), that WRN is involved in the processing of DNA, it may be required for the repair of a specific of DNA A on the ligation of DNA in from WS patients an increased of deletion at the ligation in end T.M. C. B. K. P. C. M. Martin G.M. J. 1994; Full Text PDF PubMed Google Scholar). These data may indicate a in the of the repair for the that WRN and Ku may be of a pathway from studies of Ku80 mutant and from Ku80 display such as a higher of chromosomal characterized by DNA and E. R. U. M. Genet. PubMed Scopus Google Scholar, H. G. M. P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, M.J. J. Chen E. T. A. 2000; PubMed Scopus Google Scholar). In addition, Ku80 show of premature aging, such as and cancer H. G. M. P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). both Ku80 and WRN to the of Interestingly, WRN is in the of human L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google L. H. Martin G.M. Oshima J. Exp. Cell Res. 1998; PubMed Scopus Google Scholar), the that its function may be required for a specific process. On the other hand, it is also that WRN is and in the to be it is as for factors 1999; PubMed Scopus Google Scholar). Our is to the specific process in which the WRN activity is The to this and other on the function of the WRN important on the activities involved in the process of We H. of for the Ku We are to T. for and to the of the Comai for and