Telomere-binding Protein TRF2 Binds to and Stimulates the Werner and Bloom Syndrome Helicases

Abstract

Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere maintenance including genomic instability, premature senescence, and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends. Werner syndrome is a human premature aging disorder displaying cellular defects associated with telomere maintenance including genomic instability, premature senescence, and accelerated telomere erosion. The yeast homologue of the Werner protein (WRN), Sgs1, is required for recombination-mediated lengthening of telomeres in telomerase-deficient cells. In human cells, we report that WRN co-localizes and physically interacts with the critical telomere maintenance protein TRF2. This interaction is mediated by the RecQ conserved C-terminal region of WRN. In vitro, TRF2 demonstrates high affinity for WRN and for another RecQ family member, the Bloom syndrome protein (BLM). TRF2 interaction with either WRN or BLM results in a notable stimulation of their helicase activities. Furthermore, the WRN and BLM helicases, partnered with replication protein A, actively unwind long telomeric duplex regions that are pre-bound by TRF2. These results suggest that TRF2 functions with WRN, and possibly BLM, in a common pathway at telomeric ends. Defects in members of the RecQ family of DNA helicases are responsible for three distinct human disorders: Werner syndrome (WS) 1The abbreviations used are: WS, Werner syndrome; WRN, Werner protein; TRF2, TTAGGG repeat binding factor 2; BLM, Bloom protein; RPA, replication protein A; ALT, alternative lengthening of telomeres; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; NE, nuclear extracts; ELISA, enzyme-linked immunosorbent assay; EGFP, enhanced green fluorescent protein; RQC, conserved RecQ C-terminal region; PML, promyelocytic leukemia. (1Matsumoto T. Shimamoto A. Goto M. Furuichi Y. Nat. Genet. 1997; 16: 335-336Google Scholar), Bloom syndrome (2Ellis N.A. German J. Hum. Mol. Genet. 1996; 5: 1457-1463Google Scholar), and Rothmund-Thomson syndrome (3Kitao S. Ohsugi I. Ichikawa K. Goto M. Furuichi Y. Shimamoto A. Genomics. 1998; 54: 443-452Google Scholar). All three disorders exhibit a predisposition to cancer and increased genomic instability (4Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Google Scholar). WS is of particular interest in aging research, because WS patients prematurely display aging features including gray hair, cataracts, osteoporosis, atherosclerosis, and diabetes mellitus (type II) (5Chakraverty R.K. Hickson I.D. Bioessays. 1999; 21: 286-294Google Scholar). Cells from WS patients show elevated levels of DNA deletions, translocations, and chromosomal breaks (6Fukuchi K. Martin G.M. Monnat R.J. Proc. Nat. Acad. Sci., U. S. A. 1989; 86: 5893-5897Google Scholar, 7Stefanini M. Scappaticci S. Lagomarsini P. Borroni G. Berardesca E. Nuzzo F. Mutation Res. 1989; 219: 179-185Google Scholar) and display replicative defects including an extended S-phase and premature senescence (8Martin G.M. Sprague C.A. Epstein C.J. Lab. Invest. 1970; 23: 86-92Google Scholar, 9Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Google Scholar). The gene defective in WS (10Yu C.E. Oshima J., Fu, Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Google Scholar) encodes a 167-kDa protein (WRN) that has ATPase, 3′ → 5′ helicase, and 3′ → 5′ exonuclease activities (11Gray 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: 100-103Google Scholar, 12Huang S., Li, B. Gray M.D. Oshima J. Mian I.S. Campisi J. Nat. Genet. 1998; 20: 114-116Google Scholar, 13Kamath-Loeb A.S. Shen J.C. Loeb L.A. Fry M. J. Biol. Chem. 1998; 273: 34145-34150Google Scholar). The status of the WRN protein in the cell influences not only genome integrity but also the cellular life span. The state of telomeres in the cell is also an important determinant of cellular life span. Telomeres cap and protect chromosome ends, and the consequences of telomere dysfunction include replicative senescence, apoptosis, and/or genomic instability (reviewed in Ref. 14Campisi J. Kim S. Lim C.S. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Google Scholar). Telomere dysfunction results from direct damage, defects in telomere maintenance proteins, and a progressive decline in telomere lengths that occurs with each cell division (reviewed in Ref. 15Blackburn E.H. Nature. 2000; 408: 53-56Google Scholar). Telomere-associated senescence can be bypassed in some cell lines by the expression of telomerase, which extends telomeres (14Campisi J. Kim S. Lim C.S. Rubio M. Exp. Gerontol. 2001; 36: 1619-1637Google Scholar). Evidence suggests that proper telomere function in mammalian cells is maintained by a nucleoprotein complex, together with structural features of the telomeric DNA. Telomere repeat binding factors TRF1 and TRF2 regulate telomere length, and TRF2 defects induce growth arrest and telomere end fusions (16Smogorzewska A. van Steensel B. Bianchi A. Oelmann S. Schaefer M.R. Schnapp G. De Lange T. Mol. Cell. Biol. 2000; 20: 1659-1668Google Scholar,17van Steensel B. Smogorzewska A. De Lange T. Cell. 1998; 92: 401-413Google Scholar). The TRF proteins specifically bind to duplex telomeric DNA, but not to single stranded DNA, and aid in the formation and stabilization of telomeres in a secondary structure that sequesters and protects the telomeric end (18Broccoli D. Smogorzewska A. Chong L. De Lange T. Nat. Genet. 1997; 17: 231-235Google Scholar, 19Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. De Lange T. Cell. 1999; 97: 503-514Google Scholar). Recent evidence suggests that the RecQ helicases may also function in pathways at telomeric ends. Accelerated rates of telomere shortening and defective repair at telomeres were observed in WS fibroblasts (20Kruk P.A. Rampino N.J. Bohr V.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 258-262Google Scholar,21Schulz V.P. Zakian V.A. Ogburn C.E. McKay J. Jarzebowicz A.A. Edland S.D. Martin G.M. Hum. Genet. 1996; 97: 750-754Google Scholar). Altered telomere length dynamics were reported in WS B-lymphoblastoid cell lines (22Tahara H. Tokutake Y. Maeda S. Kataoka H. Watanabe T. Satoh M. Matsumoto T. Sugawara M. Ide T. Goto M. Furuichi Y. Sugimoto M. Oncogene. 1997; 15: 1911-1920Google Scholar). Furthermore, the expression of telomerase in WS fibroblasts extended the cellular life span, rescued the premature senescent phenotype (23Wyllie F.S. Jones C.J. Skinner J.W. Haughton M.F. Wallis C. Wynford-Thomas D. Faragher R.G. Kipling D. Nat. Genet. 2000; 24: 16-17Google Scholar), and partially reversed the hypersensitivity to 4NQO (24Hisama F.M. Chen Y.H. Meyn M.S. Oshima J. Weissman S.M. Cancer Res. 2000; 60: 2372-2376Google Scholar). The molecular basis for the apparent connection between WRN and telomerase is unknown. In yeastSaccharomyces cerevisiae the RecQ homolog, Sgs1, participates in a telomerase-independent pathway for telomere lengthening (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar, 26Cohen H. Sinclair D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3174-3179Google Scholar, 27Huang P. Pryde F.E. Lester D. Maddison R.L. Borts R.H. Hickson I.D. Louis E.J. Curr. Biol. 2001; 11: 125-129Google Scholar). This mechanism for telomere maintenance, termed alternative lengthening of telomeres also in mammalian cells T.M. A. L. Nat. 1997; Scholar). cells are by long telomeres and distinct nuclear to promyelocytic which the protein telomeric DNA, proteins RPA, and proteins TRF1 and TRF2 A.A. A. Cancer Res. 1999; Scholar). WRN to with nuclear (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar), and is evidence for of the Bloom syndrome protein to Marciniak R.A. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Scholar). is not RecQ helicases in a telomere pathway in mammalian cells. report molecular evidence for the of human RecQ helicases, WRN and BLM, in a protein that functions specifically in DNA at telomeric ends. observed that protein TRF2 physically with the WRN protein in and to WRN and BLM with high affinity in In TRF2 the helicase of WRN and BLM protein for the WRN and BLM helicase together with TRF2, in telomeric DNA WRN protein a cell expression R.M. A. Gray M.D. Bohr V.A. Res. 1999; Scholar). BLM cerevisiae and R.H. Hickson I.D. Curr. Biol. 1999; Scholar). human TRF2 an a cell expression The for TRF2 expression by Lange TRF2 protein from expression a with to the TRF2 protein and L. S. De Lange T. Mol. Cell. Biol. 1992; Scholar), and the by the a The protein by by The of TRF2 by nuclear R.M. C. P. J. I. Bohr V.A. EMBO J. 2001; 20: Scholar), were with protein for at were with either of or of a at to each by at for were and with of proteins were by in for and were by and and by and cells were in and bovine serum The cells were with the expression C. P. L. P. R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. Scholar). cells were and with and in for at with PBS, the were in in at were with for at were with the were with and a in were with The gene TRF2 in the the of This used to TRF2 protein by in and a and to the with WRN protein with either or were R.M. C. P. J. I. Bohr V.A. EMBO J. 2001; 20: Scholar, C. P. L. P. R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. Scholar). were to glutathione and with either or protein in by and by of the WRN by either or and by TRF2 from were by with Li, Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; Scholar), with some were with of WRN or BLM protein to in or with of a by for at were and with of BSA, for at of TRF2 protein were and for at TRF2 and for at were and secondary and for at were and TRF2 with by with at and were for in the the at a of the for the and the of WRN or BLM by TRF2 and the were by and binding Li, Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; Scholar). The and the and duplex R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar). the but the telomeric The duplex by to The duplex by to The by the from a by by with and The WRN exonuclease duplex DNA with a 3′ J. P. Bohr V.A. J. Biol. Chem. 2001; Scholar). All DNA are were in and and DNA were in the were for at and by of and to a were and at in were a and The of DNA the of that the were in at for The DNA and protein were in the with the the proteins were at for to of the DNA with the TRF2 with the DNA for to the of the helicase with or were by the of and to a with a of to of the single stranded DNA the duplex were with R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar). This TRF2 and with the DNA and that to the were and a The of R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar). were and protein were in the the duplex the with TRF2 to the of WRN and at for with the duplex the 3′ WRN with TRF2 for to the of and at for The were by of R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar). were and were a the reported of WRN with proteins in nuclear (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar), we for a between The C-terminal of WRN the binding to proteins V.A. M. D. A. J. J. P. R. Exp. Gerontol. 2000; Scholar). we used a to for the of a C-terminal with proteins in TRF2 with the but not with the protein by with TRF2 and WRN of the that were with of the to the to the of the TRF2 interaction with with the that of the TRF2 protein for of TRF2 and WRN in we the the of human TRF2 WRN protein with TRF2 with the that of the WRN protein to either TRF2 or WRN and TRF2 from with a WRN C-terminal we were to TRF2 with a WRN because of or because the of WRN to results that a of TRF2 and WRN in the we for WRN and TRF2 in cells. In the of the may the of WRN and TRF2 in cell including (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar). the we human cell lines with a to WRN to the enhanced green fluorescent protein reported for WRN (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar), the to nuclear in the cell The of with TRF2 to WRN (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar). in cells to the with for WRN R.A. F. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1998; Scholar). that TRF2 the with not observed in cells not in a of cells reported C. P. L. P. R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. either in the and in nuclear or in nuclear In cells, of the nuclear with distinct TRF2 the of the and WRN has observed to nuclear in to some DNA and S-phase arrest S. K. Goto M. Furuichi Y. Shimamoto A. 2001; Scholar, A. M. R.M. Bohr V.A. Hickson I.D. EMBO 2000; Scholar). WRN and TRF2 may cellular which the in a common of WRN protein C. P. L. P. R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. Scholar) used to the of protein interaction with TRF2. protein that the WRN protein were The were with TRF2 and the by and not with binding to the WRN which a of the WRN exonuclease of WRN and with in a and These results that TRF2 with the WRN but also a the WRN helicase and conserved RecQ C-terminal we the in the WRN responsible for the interaction with in to the and the In only with the WRN the These that TRF2 to WRN the WRN and of interaction in the WRN and in the WRN exonuclease the direct interaction between WRN and TRF2 and to the of the protein we binding proteins TRF2 to the conserved of WRN we also another RecQ family member, the BLM for binding to TRF2. BLM encodes a protein with and helicase and with the WRN helicase P. Bohr V.A. Hickson I.D. Res. 2001; Scholar). for were by the of with WRN, or BLM by with TRF2 TRF2 an TRF2 and The of TRF2 protein to with WRN and BLM in an in the to the with This not in the of that the observed and were not mediated by DNA. In observed TRF2 to which to the of the TRF2 interaction with WRN and Furthermore, the increased a function of TRF2 for WRN and BLM and to at TRF2 and WRN and TRF2 and BLM the of the and the in were by and the apparent were the binding Li, Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; Scholar). The apparent for the and were and These are to that reported for the BLM and interaction Li, Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; Scholar). These a high affinity interaction between TRF2 and the human RecQ helicases, WRN and the TRF2 interaction with WRN and BLM their we duplex that replication and repair at telomeric ends. were to be by the WRN and BLM helicases R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar, P. Bohr V.A. Hickson I.D. Res. 2001; Scholar). The to bind TRF2 and telomeric the stranded TRF2 DNA a of the binding are required for binding (18Broccoli D. Smogorzewska A. Chong L. De Lange T. Nat. Genet. 1997; 17: 231-235Google Scholar, A. Stansel R.M. L. J.D. D. De Lange T. EMBO J. 1999; Scholar). were that not bind TRF2. the of the to a In a duplex to only telomeric and a single we for TRF2 binding to the a In were either in the or of TRF2 and were The to the and not This with which that the of TRF2 with of (18Broccoli D. Smogorzewska A. Chong L. De Lange T. Nat. Genet. 1997; 17: 231-235Google Scholar). the in the of TRF2 but in the with TRF2 in the of DNA. of DNA in the an of we the of TRF2 to bind the duplex to in the WRN and BLM helicase of TRF2 with the duplex in a of the DNA In and TRF2 the of DNA that and in the and In the of that and by TRF2 of TRF2 with the or the and These that the TRF2 protein to the duplex but not to the or the the of TRF2 to WRN and BLM we used the because can be by the WRN helicase R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar) but is not by TRF2 observed the helicase be to TRF2 with the helicase to TRF2 with the DNA. used of helicase that either stimulation or of be The helicase proteins were with TRF2 to the of and the were by WRN of of the duplex of WRN, together with of TRF2 to not the of of WRN with and in a and in the of a TRF2 A, to with WRN the helicase stimulation not stimulation at an of TRF2 to WRN stimulation not at TRF2 to WRN not observed a in which the WRN and with TRF2 in In the with the duplex and also to WRN A. R.M. D. Bohr V.A. 2000; in of TRF2, stimulation of WRN helicase observed not Furthermore, a helicase, with of TRF2 observed not These that of to TRF2, to WRN, results in a notable stimulation of the WRN helicase duplex The TRF2 interaction with WRN may be conserved RecQ helicases, results were with BLM and the duplex in to WRN, of BLM with and TRF2 in a and in the of by BLM for WRN, stimulation observed at an of TRF2 to BLM stimulation not observed at TRF2 to BLM not In results that TRF2 formation with the RecQ helicases, WRN and BLM, their the of TRF2 to the WRN exonuclease we used a duplex DNA with a 3′ end J. P. Bohr V.A. J. Biol. Chem. 2001; Scholar). The were a in the WRN exonuclease of the at the 3′ end and a of of WRN with of TRF2, to not the of exonuclease TRF2 interaction with WRN not the WRN we WRN and BLM were to unwind telomeric duplex that were pre-bound by TRF2. a of TRF2 protein to that binding to the of the In with the we reported that the WRN helicase not unwind the duplex region but that the WRN exonuclease at the end R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar). the of the human single stranded RPA, WRN helicase to unwind long that are not by WRN R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar, R.M. A. Bohr V.A. J. Biol. Chem. 1999; Scholar). we the of WRN and the that were either or pre-bound by TRF2 helicase for of WRN with either or in the of the duplex and the of because of WRN exonuclease TRF2 binding to the telomeric not the WRN exonuclease This the were a in B. In TRF2 not WRN to unwind a long duplex that WRN unwind WRN with of the duplex A, and of the TRF2 pre-bound TRF2 interaction with the not the of the helicase or the WRN exonuclease to WRN, the BLM protein not unwind the duplex region of the observed with BLM to that by which can duplex by binding single stranded of the duplex by BLM is by the of TRF2 that TRF2 is to BLM of long In the of TRF2 the not of TRF2 to the DNA to of by the of the observed for WRN, the of the BLM helicase to unwind the duplex of with the not the of the helicase In WRN and BLM partnered with helicase by TRF2. In we observed that the Werner syndrome protein partially and physically with the protein TRF2 in The interaction mediated by the conserved of WRN, which is also in RecQ helicases, including the Bloom syndrome with we observed that TRF2 has high affinity for WRN and BLM proteins in that formation of WRN and BLM with TRF2 for their helicase and in a to stimulation of Furthermore, and helicase actively long telomeric duplex regions that were pre-bound by TRF2. The TRF2 stimulation of WRN helicase to be for TRF2. proteins that also with WRN, including A. R.M. D. Bohr V.A. 2000; Scholar), J. P. Bohr V.A. J. Biol. Chem. 2001; Scholar), R.M. C. P. J. I. Bohr V.A. EMBO J. 2001; 20: Scholar), BLM C. P. L. P. R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. Scholar), and and to a helicase proteins, and BLM also bind to the WRN R.M. C. P. J. I. Bohr V.A. EMBO J. 2001; 20: Scholar, C. P. L. P. R.M. Hickson I.D. Bohr V.A. J. Biol. Chem. Scholar). The mechanism by which TRF2 WRN and BLM helicase from stimulation of interaction with WRN and BLM of long that are not by either helicase Li, Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; Scholar, R.M. A. Bohr V.A. J. Biol. Chem. 1999; Scholar), TRF2 not TRF2 not bind to single stranded DNA (18Broccoli D. Smogorzewska A. Chong L. De Lange T. Nat. Genet. 1997; 17: 231-235Google Scholar) and partially to interaction of TRF2 with WRN may the in an or may the interaction with DNA. WRN, and possibly BLM, at the in repair including the with the of the protein D. Curr. Biol. 2001; 11: Scholar) and the B. M. Lange T. Nat. Genet. 2000; Scholar) observed to to is evidence that defects in and in telomeres in mammalian cells S.M. J. Chen Li, E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar, S. H. I. F. E. P. R.A. Curr. Biol. 2001; 11: Scholar). a for WRN in repair is by a and interaction with and the increased of WS cells to A. R.M. D. Bohr V.A. 2000; S.M. S. S. Campisi J. Chen J. Biol. Chem. 2001; Scholar). to WRN, and also with TRF2 B. M. Lange T. Nat. Genet. 2000; K. D. Y. I. 2000; Scholar). repair in telomere maintenance is not but function in the cellular to Recent suggest that cells may to the state or structure of telomeres to the telomeric length E.H. Nature. 2000; 408: 53-56Google Scholar). E.H. Nature. 2000; 408: 53-56Google Scholar) has a telomeres in an and a of a that telomerase observed with mammalian telomeres by TRF2 J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. De Lange T. Cell. 1999; 97: 503-514Google Scholar). the telomeric 3′ single stranded the duplex region in a that is by a at the of J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. De Lange T. Cell. 1999; 97: 503-514Google Scholar). The in repair and including WRN, may function in the cellular to telomeric which are The telomere state that telomeres a of in the and that the of telomerase or ALT, can the telomere to the E.H. Nature. 2000; 408: 53-56Google Scholar). WS cells display an accelerated of telomere the telomere lengths of the senescent WS cells were of the fibroblasts V.P. Zakian V.A. Ogburn C.E. McKay J. Jarzebowicz A.A. Edland S.D. Martin G.M. Hum. Genet. 1996; 97: 750-754Google Scholar). the premature senescence observed in WS cells is not by telomeres but may be to telomere with the expression of telomerase to senescence in WS cells (23Wyllie F.S. Jones C.J. Skinner J.W. Haughton M.F. Wallis C. Wynford-Thomas D. Faragher R.G. Kipling D. Nat. Genet. 2000; 24: 16-17Google Scholar), for cell This suggests that telomeres in WS may a of in the are expression of telomerase may the for WRN to in a pathway at is that WRN functions in the pathway for lengthening or for in yeast (25Johnson F.B. Marciniak R.A. McVey M. Stewart S.A. Hahn W.C. Guarente L. EMBO J. 2001; 20: 905-913Google Scholar). WRN and BLM may to at The of secondary at telomeric a molecular mechanism for to an to for the of replication and the of repair and telomere lengthening that WRN and BLM unwind telomeric pre-bound by TRF2 suggests that may to that WRN helicase at and BLM, is at the of with WRN to the in and cell lines but has observed to the and with and/or in to replication and some S. K. Goto M. Furuichi Y. Shimamoto A. 2001; Scholar, A. M. R.M. Bohr V.A. Hickson I.D. EMBO 2000; Scholar). The interaction we observed between TRF2 and WRN suggests that TRF2 has the to WRN to the telomeres WRN has the BLM is the (5Chakraverty R.K. Hickson I.D. Bioessays. 1999; 21: 286-294Google Scholar) and may be by TRF2 to specifically in the of telomeric DNA that and helicase the to unwind long telomeric in because to unwind to long Li, Hickson I.D. Bohr V.A. J. Biol. Chem. 2000; Scholar, R.M. A. Bohr V.A. J. Biol. Chem. 1999; Scholar), and their is not by TRF2 or R.M. Bohr V.A. J. Biol. Chem. 2001; Scholar). In TRF1 interaction with telomeric DNA reported to the of DNA A. Stansel R.M. L. J.D. D. De Lange T. EMBO J. 1999; Scholar). the RecQ helicases may also function to the and to some DNA at telomeric ends. The of WRN, and BLM, at telomeric to be for helicase also and for and and for critical of the also members of the of for