Two Independent Regions of Human Telomerase Reverse Transcriptase Are Important for Its Oligomerization and Telomerase Activity
2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês
10.1074/jbc.m111068200
ISSN1083-351X
AutoresKuniaki Arai, Kenkichi Masutomi, Shilagardy Khurts, Shuichi Kaneko, Kenichi Kobayashi, Seishi Murakami,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoHuman telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, contains motifs conserved among reverse transcriptases. Several nucleic acid-dependent polymerases that share a “fingers, palm, and thumb substructure” were shown to oligomerize. Here we demonstrate that hTERT also has this ability using partially purified recombinant hTERTs and mammalian cells co-expressing differently tagged hTERTs. Human template RNA (hTR), by contrast, has no effect on the structural oligomerization of hTERTs. Therefore, hTERT has an intrinsic ability of oligomerization in the absence of hTR. We identified two separate regions as essential for the oligomerization. The regions, amino acids 301–538 (amino-terminal region) and amino acids 914–928 (carboxyl-terminal region), are outside the fingers and palm substructure covering motif T to D and interact with each otherin vivo. A substituted mutant of hTERT, hTERT-D712A-V713I, which was reported as a dominant negative form of hTERT, bound to the wild-type hTERT and inhibited its telomerase activity transiently expressed in telomerase-negative finite normal human fibroblast. The truncated forms of hTERT containing the binding region to the wild-type hTERT partially inhibited the telomerase activity, probably by preventing the wild-type hTERT from forming an oligomer. Taken together, the oligomerization of hTERT is an important step for telomerase activity. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, contains motifs conserved among reverse transcriptases. Several nucleic acid-dependent polymerases that share a “fingers, palm, and thumb substructure” were shown to oligomerize. Here we demonstrate that hTERT also has this ability using partially purified recombinant hTERTs and mammalian cells co-expressing differently tagged hTERTs. Human template RNA (hTR), by contrast, has no effect on the structural oligomerization of hTERTs. Therefore, hTERT has an intrinsic ability of oligomerization in the absence of hTR. We identified two separate regions as essential for the oligomerization. The regions, amino acids 301–538 (amino-terminal region) and amino acids 914–928 (carboxyl-terminal region), are outside the fingers and palm substructure covering motif T to D and interact with each otherin vivo. A substituted mutant of hTERT, hTERT-D712A-V713I, which was reported as a dominant negative form of hTERT, bound to the wild-type hTERT and inhibited its telomerase activity transiently expressed in telomerase-negative finite normal human fibroblast. The truncated forms of hTERT containing the binding region to the wild-type hTERT partially inhibited the telomerase activity, probably by preventing the wild-type hTERT from forming an oligomer. Taken together, the oligomerization of hTERT is an important step for telomerase activity. telomerase reverse transcriptase template RNA human RNA-dependent RNA polymerase hemagglutinin glutathione S-transferase n-nonanoyl-N-methylglucomide telomerase repeat amplification protocol enzyme-linked immunosorbent assay amino acid(s) human immunodeficiency virus Telomeres are specialized structures positioned at the ends of linear eukaryotic chromosomes that provide a mechanism for maintaining chromosome length and stability. The termini of telomeric DNA cannot be fully replicated by the conventional replication machinery. Telomerase, a ribonucleoprotein complex composed of template RNA and several proteins, elongates telomeres as one means of end replication (1McEachern M.J. Krauskopf A. Blackburn E.H. Annu. Rev. Genet. 2000; 34: 331-358Crossref PubMed Scopus (603) Google Scholar). Telomerase reverse transcriptase (TERT1), the catalytic subunit of telomerase, is a specific type of reverse transcriptase that forms stable complexes with template RNA (TR) (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar). Human TERT is the rate-limiting factor for telomerase activity both biologically and biochemically (3Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar, 4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Introduction of hTERT into normal human primary cells overcomes senescence and extends their lifespan (3Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar, 5Nakayama J. Tahara H. Tahara E. Saito M. Ito K. Nakamura H. Nakanishi T. Tahara E. Ide T. Ishikawa F. Nat. Genet. 1998; 18: 65-68Crossref PubMed Scopus (584) Google Scholar). We recently reported that hTERT and hTR, are the minimum components required for telomerase activity reconstituted in vitro with purified forms (4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).TERT is part of a large family of nucleic acid-dependent nucleic acid polymerases that share a “ fingers, palm, and thumb substructure” (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar, 6Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 7Sousa R. Trends Biochem. Sci. 1996; 21: 186-190Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 8Bressanelli S. Tomei L. Roussel A. Incitti I. Vitale R.L. De Mathieu M. Francesco R. Rey F.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13034-13039Crossref PubMed Scopus (542) Google Scholar). Human TERT contains several motifs conserved among many reverse transcriptases, and additional motifs conserved only among TERTs from species ranging from budding yeasts to humans (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar). Some polymerases that share a fingers, palm, and thumb substructure, such as HIV reverse transcriptase, polio RNA-dependent RNA polymerases (RdRP), and hepatitis C virus RdRP, oligomerize (12Muller B. Restle T. Weiss S. Gautel M. Sczakiel G. Goody R.S. J. Biol. Chem. 1989; 264: 13975-13978Abstract Full Text PDF PubMed Google Scholar, 13Restle T. Muller B. Goody R.S. J. Biol. Chem. 1990; 265: 8986-8988Abstract Full Text PDF PubMed Google Scholar, 14Tachedjian G. Aronson H.E. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6334-6339Crossref PubMed Scopus (54) Google Scholar, 15Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar, 16Qin W. Luo H. Nomura T. Hayashi N. Yamashita T. Murakami S. J. Biol. Chem. 2002; 277: 2132-2137Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Oligomerization in these enzymes induces conformational changes, which provide active or open forms that are essential for catalytic functions.In Saccharomyces cerevisiae, telomerase forms an active multimer in vivo that may contain two active sites. This suggests that Est2p can oligomerize (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar). Recently, one group reported that the human telomerase complex also forms a homodimer that contains two template RNA molecules (18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar), and another group found that two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity (19Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (119) Google Scholar). These results indicated that just one hTERT molecule and one hTR molecule alone could not reconstitute telomerase activity. In other words, hTERT and hTR molecules must form a multimer and reconstitute telomerase activity by working together. However, it was not clear whether disruptions to the oligomeric formation of hTERT reduce telomerase activity.Here we demonstrate that two independent regions outside of motifs T to D have an important role in the oligomeric interaction of hTERTin vitro using purified recombinant hTERT in the absence of hTR, and in mammalian cells transiently co-expressing various tagged hTERTs. These two independent regions can interact. We also demonstrate that catalytically inactive truncated forms of hTERT, which contain the binding region can inhibit telomerase activity of the wild-type hTERT.DISCUSSIONTERT is a unique enzyme among a family of nucleic acid-dependent polymerases harboring a fingers, palm, and thumb substructure, because it forms a tight complex with template RNA for the activity (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 24Xiong Y. Eickbush T.H. EMBO J. 1990; 9: 3353-3362Crossref PubMed Scopus (1120) Google Scholar). Its long amino- and carboxyl-terminal parts outside of the fingers and palm (aa 525–928) might retain TERT-specific functions (11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar, 22Counter C.M. Hahn W.C. Wei W. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Crossref PubMed Scopus (558) Google Scholar, 25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 27Seimiya H. Sawada H. Muramatsu Y. Shimizu M. Ohko K. Yamane K. Tsuruo T. EMBO J. 2000; 19: 2652-2661Crossref PubMed Scopus (253) Google Scholar, 28Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar), because these parts are somewhat conserved only among TERTs (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar). We previously reported that hTERT and hTR are the minimum components required for telomerase activity when telomerase is reconstituted in vitro with two purified components (4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). During this study we found that the catalytic activity of the purified hTERT was not detectable when concentrations of hTERT were low in the assay reaction (data not shown). This concentration dependence of hTERT reminded us of the template switching of telomerase previously reported in S. cerevisiae (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar) and the oligomeric interactions of poliovirus (15Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar) and hepatitis C virus RdRPs (29Qin W. Yamashita T. Shirota Y. Lin Y. Wei W. Murakami S. Hepatology. 2001; 33: 728-737Crossref PubMed Scopus (54) Google Scholar). Two groups recently reported the oligomeric role of telomerase using the different methods (18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar, 19Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (119) Google Scholar). However, it was not clear whether TERT forms multimer intrinsically or with help of template RNA. Here we show that the homomeric interaction of hTERT in vitro with partially purified differently tagged-hTERTs. Human TR seems to have no effect on the structural oligomerization of hTERT (Fig. 1 C), and RNase treatment did not affect the homomeric interaction in vivo (Fig.2 A) and in vitro (data not shown). These results indicate that hTERT has an intrinsic ability to oligomerize in the absence of intact hTR.Two separate regions of hTERT (aa 301–538 and aa 914–1132) can be mapped to bind the wild-type hTERT in vitro and in vivo. This result may be consistent with a previous report (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar) in which some combinations of two different truncated hTERT mutants defective in telomerase activity reconstituted telomerase activity. Two regions we mapped are outside the fingers and palm substructure covering motifs T to D. The amino-terminal fragment, aa 301–538, overlaps the region important for hTR binding (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 30Bachand F. Autexier C. Mol. Cell. Biol. 2001; 21: 1888-1897Crossref PubMed Scopus (105) Google Scholar). The carboxyl-terminal fragment, aa 914–1132, includes motif E and a putative thumb. The two separate regions can bind each other, but no homologous interaction of aa 301–538 or aa 914–1132 was detected (Fig. 4 A and data not shown). The result seems to support a model that the homomeric interaction of hTERT occurs in a “head to tail” fashion. Our result cannot explain the previous result that the amino-terminal region (aa 1–300) and some truncated mutants (such as aa 301–1132) reconstituted telomerase activity (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar). The region (aa 1–300) critical for telomerase activity (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar) may be not essential for the oligomerization but essential to interact with one or more critical factors such as hTR-binding proteins or Hsp90, which is recruited to telomerase by hTR or hTERT.Functional oligomeric formations of telomerase have been proposed since two functional template RNAs in the telomerase complex having two active sites were found in S. cerevisiae and in humans (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar,18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar). In our experiments, the substituted mutant hTERT-D712A-V713I, bound to the wild-type hTERT in vivo and in vitro(data not shown) and inhibited its telomerase activity (Fig. 6), suggesting that oligomeric formation of the wild-type and the mutant hTERTs is the reason of the inhibition. The D712A or D712A-V713I mutants at the VDV sequence, which is critical for substrate binding, have been previously described as dominant negative mutants that eliminate endogenous telomerase activity and cause telomere shortening and cell senescence due to lack of telomerase activity (23Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (936) Google Scholar, 31Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 15: 2388-2399Crossref Scopus (557) Google Scholar). However, the inhibitory effect of the mutant on the wild-type hTERT was not severe when transiently co-expressed in the telomerase-negative cells even if the amount of plasmid of the mutant was more than 10 times higher than the wild-hTERT (Fig. 6). The result seems to be consistent with the previous report by Beattie et al. (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar) who observed a partial restoration of telomerase activity in the presence of hTERT-D712A and the truncated hTERTs harboring the intact pocket for active center but missing the amino-terminal region. These results suggest that the mutants defective in substrate binding (D712A or D712A-V713I) are not dominant negative enzymatically when these mutants oligomerized with the wild-type hTERT, although reconstituted telomerase activity of oligomers consisting of the wild-type and D712A or D712A-V713I hTERTs seems to be much weaker than that of the wild-type oligomer (Fig. 6) (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar). The apparent dominant negative phenotype of D712A or D712A-V713I in the previous reports may be explained by a huge difference in expression levels between endogenous hTERT and the ectopically expressed mutant hTERT, which may squelch out one or more critical factors for telomerase activity.We expected that the truncated mutants, which have the hTERT-binding regions, would exhibit strong inhibitory effects on the wild-type hTERT by competing oligomerization of the wild-type hTERT. Rather weak inhibitory effects of the amino-terminal and the carboxyl-terminal binding regions may be due to weaker binding abilities to the wild-type hTERT or inefficient recruitment of the proteins to the wild-type hTERT.Telomere maintenance is essential to the replicative potential of malignant cells, and inhibition of telomerase leads to telomere shorting and cessation of unrestrained proliferation (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 23Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (936) Google Scholar,31Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 15: 2388-2399Crossref Scopus (557) Google Scholar, 32Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6493) Google Scholar, 33Ramakrishnan S. Eppenberger U. Mueller H. Shinkai Y. Narayanan R. Cancer Res. 1998; 58: 622-625PubMed Google Scholar). The intrinsic property of hTERT to oligomerize may be an additional target to design specific inhibitors of telomerase. This strategy has been applied to HIV reverse transcriptase (14Tachedjian G. Aronson H.E. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6334-6339Crossref PubMed Scopus (54) Google Scholar,34Harris D. Lee R. Misra H.S. Pandey P.K. Pandey V.N. Biochemistry. 1998; 37: 5903-5908Crossref PubMed Scopus (44) Google Scholar, 35Divita G. Restle T. Goody R.S. Chermann J.C. Baillon J.G. J. Biol. Chem. 1994; 269: 13080-13083Abstract Full Text PDF PubMed Google Scholar, 36Morris M.C. Robert-Hebmann V. Chaloin L. Mery J. Heitz F. Devaux C. Goody R.S. Divita G. J. Biol. Chem. 1999; 274: 24941-24946Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Telomeres are specialized structures positioned at the ends of linear eukaryotic chromosomes that provide a mechanism for maintaining chromosome length and stability. The termini of telomeric DNA cannot be fully replicated by the conventional replication machinery. Telomerase, a ribonucleoprotein complex composed of template RNA and several proteins, elongates telomeres as one means of end replication (1McEachern M.J. Krauskopf A. Blackburn E.H. Annu. Rev. Genet. 2000; 34: 331-358Crossref PubMed Scopus (603) Google Scholar). Telomerase reverse transcriptase (TERT1), the catalytic subunit of telomerase, is a specific type of reverse transcriptase that forms stable complexes with template RNA (TR) (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar). Human TERT is the rate-limiting factor for telomerase activity both biologically and biochemically (3Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar, 4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Introduction of hTERT into normal human primary cells overcomes senescence and extends their lifespan (3Bodnar A.G. Ouellette M. Frolkis M. Holt S.E. Chiu C.P. Morin G.B. Harley C.B. Shay J.W. Lichtsteiner S. Wright W.E. Science. 1998; 279: 349-352Crossref PubMed Scopus (4067) Google Scholar, 5Nakayama J. Tahara H. Tahara E. Saito M. Ito K. Nakamura H. Nakanishi T. Tahara E. Ide T. Ishikawa F. Nat. Genet. 1998; 18: 65-68Crossref PubMed Scopus (584) Google Scholar). We recently reported that hTERT and hTR, are the minimum components required for telomerase activity reconstituted in vitro with purified forms (4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). TERT is part of a large family of nucleic acid-dependent nucleic acid polymerases that share a “ fingers, palm, and thumb substructure” (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar, 6Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar, 7Sousa R. Trends Biochem. Sci. 1996; 21: 186-190Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 8Bressanelli S. Tomei L. Roussel A. Incitti I. Vitale R.L. De Mathieu M. Francesco R. Rey F.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13034-13039Crossref PubMed Scopus (542) Google Scholar). Human TERT contains several motifs conserved among many reverse transcriptases, and additional motifs conserved only among TERTs from species ranging from budding yeasts to humans (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar). Some polymerases that share a fingers, palm, and thumb substructure, such as HIV reverse transcriptase, polio RNA-dependent RNA polymerases (RdRP), and hepatitis C virus RdRP, oligomerize (12Muller B. Restle T. Weiss S. Gautel M. Sczakiel G. Goody R.S. J. Biol. Chem. 1989; 264: 13975-13978Abstract Full Text PDF PubMed Google Scholar, 13Restle T. Muller B. Goody R.S. J. Biol. Chem. 1990; 265: 8986-8988Abstract Full Text PDF PubMed Google Scholar, 14Tachedjian G. Aronson H.E. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6334-6339Crossref PubMed Scopus (54) Google Scholar, 15Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar, 16Qin W. Luo H. Nomura T. Hayashi N. Yamashita T. Murakami S. J. Biol. Chem. 2002; 277: 2132-2137Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Oligomerization in these enzymes induces conformational changes, which provide active or open forms that are essential for catalytic functions. In Saccharomyces cerevisiae, telomerase forms an active multimer in vivo that may contain two active sites. This suggests that Est2p can oligomerize (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar). Recently, one group reported that the human telomerase complex also forms a homodimer that contains two template RNA molecules (18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar), and another group found that two separate, catalytically inactive TERT proteins can complement each other in trans to reconstitute catalytic activity (19Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (119) Google Scholar). These results indicated that just one hTERT molecule and one hTR molecule alone could not reconstitute telomerase activity. In other words, hTERT and hTR molecules must form a multimer and reconstitute telomerase activity by working together. However, it was not clear whether disruptions to the oligomeric formation of hTERT reduce telomerase activity. Here we demonstrate that two independent regions outside of motifs T to D have an important role in the oligomeric interaction of hTERTin vitro using purified recombinant hTERT in the absence of hTR, and in mammalian cells transiently co-expressing various tagged hTERTs. These two independent regions can interact. We also demonstrate that catalytically inactive truncated forms of hTERT, which contain the binding region can inhibit telomerase activity of the wild-type hTERT. DISCUSSIONTERT is a unique enzyme among a family of nucleic acid-dependent polymerases harboring a fingers, palm, and thumb substructure, because it forms a tight complex with template RNA for the activity (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 24Xiong Y. Eickbush T.H. EMBO J. 1990; 9: 3353-3362Crossref PubMed Scopus (1120) Google Scholar). Its long amino- and carboxyl-terminal parts outside of the fingers and palm (aa 525–928) might retain TERT-specific functions (11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar, 22Counter C.M. Hahn W.C. Wei W. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Crossref PubMed Scopus (558) Google Scholar, 25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 27Seimiya H. Sawada H. Muramatsu Y. Shimizu M. Ohko K. Yamane K. Tsuruo T. EMBO J. 2000; 19: 2652-2661Crossref PubMed Scopus (253) Google Scholar, 28Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar), because these parts are somewhat conserved only among TERTs (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar). We previously reported that hTERT and hTR are the minimum components required for telomerase activity when telomerase is reconstituted in vitro with two purified components (4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). During this study we found that the catalytic activity of the purified hTERT was not detectable when concentrations of hTERT were low in the assay reaction (data not shown). This concentration dependence of hTERT reminded us of the template switching of telomerase previously reported in S. cerevisiae (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar) and the oligomeric interactions of poliovirus (15Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar) and hepatitis C virus RdRPs (29Qin W. Yamashita T. Shirota Y. Lin Y. Wei W. Murakami S. Hepatology. 2001; 33: 728-737Crossref PubMed Scopus (54) Google Scholar). Two groups recently reported the oligomeric role of telomerase using the different methods (18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar, 19Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (119) Google Scholar). However, it was not clear whether TERT forms multimer intrinsically or with help of template RNA. Here we show that the homomeric interaction of hTERT in vitro with partially purified differently tagged-hTERTs. Human TR seems to have no effect on the structural oligomerization of hTERT (Fig. 1 C), and RNase treatment did not affect the homomeric interaction in vivo (Fig.2 A) and in vitro (data not shown). These results indicate that hTERT has an intrinsic ability to oligomerize in the absence of intact hTR.Two separate regions of hTERT (aa 301–538 and aa 914–1132) can be mapped to bind the wild-type hTERT in vitro and in vivo. This result may be consistent with a previous report (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar) in which some combinations of two different truncated hTERT mutants defective in telomerase activity reconstituted telomerase activity. Two regions we mapped are outside the fingers and palm substructure covering motifs T to D. The amino-terminal fragment, aa 301–538, overlaps the region important for hTR binding (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 30Bachand F. Autexier C. Mol. Cell. Biol. 2001; 21: 1888-1897Crossref PubMed Scopus (105) Google Scholar). The carboxyl-terminal fragment, aa 914–1132, includes motif E and a putative thumb. The two separate regions can bind each other, but no homologous interaction of aa 301–538 or aa 914–1132 was detected (Fig. 4 A and data not shown). The result seems to support a model that the homomeric interaction of hTERT occurs in a “head to tail” fashion. Our result cannot explain the previous result that the amino-terminal region (aa 1–300) and some truncated mutants (such as aa 301–1132) reconstituted telomerase activity (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar). The region (aa 1–300) critical for telomerase activity (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar) may be not essential for the oligomerization but essential to interact with one or more critical factors such as hTR-binding proteins or Hsp90, which is recruited to telomerase by hTR or hTERT.Functional oligomeric formations of telomerase have been proposed since two functional template RNAs in the telomerase complex having two active sites were found in S. cerevisiae and in humans (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar,18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar). In our experiments, the substituted mutant hTERT-D712A-V713I, bound to the wild-type hTERT in vivo and in vitro(data not shown) and inhibited its telomerase activity (Fig. 6), suggesting that oligomeric formation of the wild-type and the mutant hTERTs is the reason of the inhibition. The D712A or D712A-V713I mutants at the VDV sequence, which is critical for substrate binding, have been previously described as dominant negative mutants that eliminate endogenous telomerase activity and cause telomere shortening and cell senescence due to lack of telomerase activity (23Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (936) Google Scholar, 31Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 15: 2388-2399Crossref Scopus (557) Google Scholar). However, the inhibitory effect of the mutant on the wild-type hTERT was not severe when transiently co-expressed in the telomerase-negative cells even if the amount of plasmid of the mutant was more than 10 times higher than the wild-hTERT (Fig. 6). The result seems to be consistent with the previous report by Beattie et al. (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar) who observed a partial restoration of telomerase activity in the presence of hTERT-D712A and the truncated hTERTs harboring the intact pocket for active center but missing the amino-terminal region. These results suggest that the mutants defective in substrate binding (D712A or D712A-V713I) are not dominant negative enzymatically when these mutants oligomerized with the wild-type hTERT, although reconstituted telomerase activity of oligomers consisting of the wild-type and D712A or D712A-V713I hTERTs seems to be much weaker than that of the wild-type oligomer (Fig. 6) (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar). The apparent dominant negative phenotype of D712A or D712A-V713I in the previous reports may be explained by a huge difference in expression levels between endogenous hTERT and the ectopically expressed mutant hTERT, which may squelch out one or more critical factors for telomerase activity.We expected that the truncated mutants, which have the hTERT-binding regions, would exhibit strong inhibitory effects on the wild-type hTERT by competing oligomerization of the wild-type hTERT. Rather weak inhibitory effects of the amino-terminal and the carboxyl-terminal binding regions may be due to weaker binding abilities to the wild-type hTERT or inefficient recruitment of the proteins to the wild-type hTERT.Telomere maintenance is essential to the replicative potential of malignant cells, and inhibition of telomerase leads to telomere shorting and cessation of unrestrained proliferation (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 23Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (936) Google Scholar,31Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 15: 2388-2399Crossref Scopus (557) Google Scholar, 32Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6493) Google Scholar, 33Ramakrishnan S. Eppenberger U. Mueller H. Shinkai Y. Narayanan R. Cancer Res. 1998; 58: 622-625PubMed Google Scholar). The intrinsic property of hTERT to oligomerize may be an additional target to design specific inhibitors of telomerase. This strategy has been applied to HIV reverse transcriptase (14Tachedjian G. Aronson H.E. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6334-6339Crossref PubMed Scopus (54) Google Scholar,34Harris D. Lee R. Misra H.S. Pandey P.K. Pandey V.N. Biochemistry. 1998; 37: 5903-5908Crossref PubMed Scopus (44) Google Scholar, 35Divita G. Restle T. Goody R.S. Chermann J.C. Baillon J.G. J. Biol. Chem. 1994; 269: 13080-13083Abstract Full Text PDF PubMed Google Scholar, 36Morris M.C. Robert-Hebmann V. Chaloin L. Mery J. Heitz F. Devaux C. Goody R.S. Divita G. J. Biol. Chem. 1999; 274: 24941-24946Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). TERT is a unique enzyme among a family of nucleic acid-dependent polymerases harboring a fingers, palm, and thumb substructure, because it forms a tight complex with template RNA for the activity (2Nugent C.I. Lundblad V. Genes Dev. 1998; 12: 1073-1085Crossref PubMed Scopus (398) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 24Xiong Y. Eickbush T.H. EMBO J. 1990; 9: 3353-3362Crossref PubMed Scopus (1120) Google Scholar). Its long amino- and carboxyl-terminal parts outside of the fingers and palm (aa 525–928) might retain TERT-specific functions (11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar, 22Counter C.M. Hahn W.C. Wei W. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Crossref PubMed Scopus (558) Google Scholar, 25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 27Seimiya H. Sawada H. Muramatsu Y. Shimizu M. Ohko K. Yamane K. Tsuruo T. EMBO J. 2000; 19: 2652-2661Crossref PubMed Scopus (253) Google Scholar, 28Zhu J. Wang H. Bishop J.M. Blackburn E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3723-3728Crossref PubMed Scopus (359) Google Scholar), because these parts are somewhat conserved only among TERTs (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 11Xia J. Peng Y. Mian I.S. Lue N.F. Mol. Cell. Biol. 2000; 20: 5196-5207Crossref PubMed Scopus (121) Google Scholar). We previously reported that hTERT and hTR are the minimum components required for telomerase activity when telomerase is reconstituted in vitro with two purified components (4Masutomi K. Kaneko S. Hayashi H. Yamashita T. Shirota Y. Kobayashi K. Murakami S. J. Biol. Chem. 2000; 275: 22568-22573Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). During this study we found that the catalytic activity of the purified hTERT was not detectable when concentrations of hTERT were low in the assay reaction (data not shown). This concentration dependence of hTERT reminded us of the template switching of telomerase previously reported in S. cerevisiae (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar) and the oligomeric interactions of poliovirus (15Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar) and hepatitis C virus RdRPs (29Qin W. Yamashita T. Shirota Y. Lin Y. Wei W. Murakami S. Hepatology. 2001; 33: 728-737Crossref PubMed Scopus (54) Google Scholar). Two groups recently reported the oligomeric role of telomerase using the different methods (18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar, 19Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Cell. Biol. 2001; 21: 6151-6160Crossref PubMed Scopus (119) Google Scholar). However, it was not clear whether TERT forms multimer intrinsically or with help of template RNA. Here we show that the homomeric interaction of hTERT in vitro with partially purified differently tagged-hTERTs. Human TR seems to have no effect on the structural oligomerization of hTERT (Fig. 1 C), and RNase treatment did not affect the homomeric interaction in vivo (Fig.2 A) and in vitro (data not shown). These results indicate that hTERT has an intrinsic ability to oligomerize in the absence of intact hTR. Two separate regions of hTERT (aa 301–538 and aa 914–1132) can be mapped to bind the wild-type hTERT in vitro and in vivo. This result may be consistent with a previous report (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar) in which some combinations of two different truncated hTERT mutants defective in telomerase activity reconstituted telomerase activity. Two regions we mapped are outside the fingers and palm substructure covering motifs T to D. The amino-terminal fragment, aa 301–538, overlaps the region important for hTR binding (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar, 30Bachand F. Autexier C. Mol. Cell. Biol. 2001; 21: 1888-1897Crossref PubMed Scopus (105) Google Scholar). The carboxyl-terminal fragment, aa 914–1132, includes motif E and a putative thumb. The two separate regions can bind each other, but no homologous interaction of aa 301–538 or aa 914–1132 was detected (Fig. 4 A and data not shown). The result seems to support a model that the homomeric interaction of hTERT occurs in a “head to tail” fashion. Our result cannot explain the previous result that the amino-terminal region (aa 1–300) and some truncated mutants (such as aa 301–1132) reconstituted telomerase activity (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar). The region (aa 1–300) critical for telomerase activity (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar, 26Armbruster B. Banik S.R.S. Guo C. Smith C.A. Counter M.C. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar) may be not essential for the oligomerization but essential to interact with one or more critical factors such as hTR-binding proteins or Hsp90, which is recruited to telomerase by hTR or hTERT. Functional oligomeric formations of telomerase have been proposed since two functional template RNAs in the telomerase complex having two active sites were found in S. cerevisiae and in humans (17Prescott J. Blackburn E.H. Genes Dev. 1997; 11: 2790-2800Crossref PubMed Scopus (145) Google Scholar,18Wenz C. Enenkel B. Amacker M. Kelleher C. Damm K. Lingner J. EMBO J. 2001; 20: 3526-3534Crossref PubMed Scopus (133) Google Scholar). In our experiments, the substituted mutant hTERT-D712A-V713I, bound to the wild-type hTERT in vivo and in vitro(data not shown) and inhibited its telomerase activity (Fig. 6), suggesting that oligomeric formation of the wild-type and the mutant hTERTs is the reason of the inhibition. The D712A or D712A-V713I mutants at the VDV sequence, which is critical for substrate binding, have been previously described as dominant negative mutants that eliminate endogenous telomerase activity and cause telomere shortening and cell senescence due to lack of telomerase activity (23Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (936) Google Scholar, 31Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 15: 2388-2399Crossref Scopus (557) Google Scholar). However, the inhibitory effect of the mutant on the wild-type hTERT was not severe when transiently co-expressed in the telomerase-negative cells even if the amount of plasmid of the mutant was more than 10 times higher than the wild-hTERT (Fig. 6). The result seems to be consistent with the previous report by Beattie et al. (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar) who observed a partial restoration of telomerase activity in the presence of hTERT-D712A and the truncated hTERTs harboring the intact pocket for active center but missing the amino-terminal region. These results suggest that the mutants defective in substrate binding (D712A or D712A-V713I) are not dominant negative enzymatically when these mutants oligomerized with the wild-type hTERT, although reconstituted telomerase activity of oligomers consisting of the wild-type and D712A or D712A-V713I hTERTs seems to be much weaker than that of the wild-type oligomer (Fig. 6) (25Beattie T.L. Zhou W. Robinson M.O. Harrington L. Mol. Biol. Cell. 2000; 11: 3329-3340Crossref PubMed Scopus (77) Google Scholar). The apparent dominant negative phenotype of D712A or D712A-V713I in the previous reports may be explained by a huge difference in expression levels between endogenous hTERT and the ectopically expressed mutant hTERT, which may squelch out one or more critical factors for telomerase activity. We expected that the truncated mutants, which have the hTERT-binding regions, would exhibit strong inhibitory effects on the wild-type hTERT by competing oligomerization of the wild-type hTERT. Rather weak inhibitory effects of the amino-terminal and the carboxyl-terminal binding regions may be due to weaker binding abilities to the wild-type hTERT or inefficient recruitment of the proteins to the wild-type hTERT. Telomere maintenance is essential to the replicative potential of malignant cells, and inhibition of telomerase leads to telomere shorting and cessation of unrestrained proliferation (9Meyerson M. Counter C.M. Eaton E.N. Ellisen L.W. Steiner P. Caddle S.D. Ziaugra L. Beijersbergen R.L. Davidoff M.J. Liu Q. Bacchetti S. Haber D.A. Weinberg R.A. Cell. 1997; 90: 785-795Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar, 10Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2051) Google Scholar, 23Hahn W.C. Stewart S.A. Brooks M.W. York S.G. Eaton E. Kurachi A. Beijersbergen R.L. Knoll J.H. Meyerson M. Weinberg R.A. Nat. Med. 1999; 5: 1164-1170Crossref PubMed Scopus (936) Google Scholar,31Zhang X. Mar V. Zhou W. Harrington L. Robinson M.O. Genes Dev. 1999; 15: 2388-2399Crossref Scopus (557) Google Scholar, 32Kim N.W. Piatyszek M.A. Prowse K.R. Harley C.B. West M.D. Ho P.L. Coviello G.M. Wright W.E. Weinrich S.L. Shay J.W. Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6493) Google Scholar, 33Ramakrishnan S. Eppenberger U. Mueller H. Shinkai Y. Narayanan R. Cancer Res. 1998; 58: 622-625PubMed Google Scholar). The intrinsic property of hTERT to oligomerize may be an additional target to design specific inhibitors of telomerase. This strategy has been applied to HIV reverse transcriptase (14Tachedjian G. Aronson H.E. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6334-6339Crossref PubMed Scopus (54) Google Scholar,34Harris D. Lee R. Misra H.S. Pandey P.K. Pandey V.N. Biochemistry. 1998; 37: 5903-5908Crossref PubMed Scopus (44) Google Scholar, 35Divita G. Restle T. Goody R.S. Chermann J.C. Baillon J.G. J. Biol. Chem. 1994; 269: 13080-13083Abstract Full Text PDF PubMed Google Scholar, 36Morris M.C. Robert-Hebmann V. Chaloin L. Mery J. Heitz F. Devaux C. Goody R.S. Divita G. J. Biol. Chem. 1999; 274: 24941-24946Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We are grateful to W. Hahn, L. Harrington, N. Hayashi, M. Hirano, and W. Qin for encouraging discussions, and to F. Momoshima, M. Yasukawa, and K. Kuwabara for technical assistance.
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