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• W1997963513 abstract "Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance. Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance. Telomeres are structures at the ends of linear eukaryotic chromosomes that prevent DNA end-initiated recombination, exonucleolytic DNA degradation, and replication-associated terminal recession. In the absence of an active mechanism to maintain telomere length, the telomere repeat number decreases with each replicative cell cycle. Telomere attrition is associated with genome instability, cell cycle arrest, and senescence or apoptosis. Telomeric DNA is composed of double-stranded tandem repeat sequences (5′-TTAGGG-3′ in human and mouse) followed by a short ssDNA 2The abbreviations used are: ssDNAsingle-stranded DNAdsDNAdouble-stranded DNARTSRothmund-Thomson syndromeKDknockdownSCRscrambledco-IPco-immunoprecipitationTIFtelomere dysfunction-induced fociT-SCEtelomere sister chromatid exchangeDL1telomeric D-loopDL48-oxoguanine-containing D-loopDLmxnon-telomeric D-loop. 3′-overhang (1Smogorzewska A. de Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (660) Google Scholar). In mammals, telomeric DNA is believed to exist as a D-loop, formed by invasion of the 3′-overhang into telomeric dsDNA (2Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar). Telomere-binding proteins, known as shelterin, and associated proteins stabilize the D-loop structure (3de Lange T. Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar). Among the shelterin proteins, POT1 (protection of telomeres 1) binds to single-stranded telomeric DNA (4Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar), and TRF1 and TRF2 (telomere repeat-binding factors 1 and 2) bind to duplex telomeric sequences (5Palm W. de Lange T. Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1384) Google Scholar). single-stranded DNA double-stranded DNA Rothmund-Thomson syndrome knockdown scrambled co-immunoprecipitation telomere dysfunction-induced foci telomere sister chromatid exchange telomeric D-loop 8-oxoguanine-containing D-loop non-telomeric D-loop. The DNA replication machinery is unable to replicate DNA ends, and as cells proliferate telomeric DNA can be lost. This terminal DNA loss is compensated for by telomerase, a reverse transcriptase that adds TTAGGG repeats at the 3′-ends of chromosomal DNA (6Greider C.W. Blackburn E.H. Cell. 1985; 43: 405-413Abstract Full Text PDF PubMed Scopus (2588) Google Scholar). The bulk of telomeric DNA is replicated by the semiconservative DNA replication machinery. The highly repetitive G-rich sequences at the ends of telomeres can adopt unusual DNA secondary structures, such as G-quadruplexes (7Verdun R.E. Karlseder J. Nature. 2007; 447: 924-931Crossref PubMed Scopus (379) Google Scholar) and D-loops (8Verdun R.E. Karlseder J. Cell. 2006; 127: 709-720Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar), which must be resolved prior to replication. Further, telomeres were identified as fragile sites, which are known to form in response to replication stress (9Martínez P. Thanasoula M. Muñoz P. Liao C. Tejera A. McNees C. Flores J.M. Fernández-Capetillo O. Tarsounas M. Blasco M.A. Genes Dev. 2009; 23: 2060-2075Crossref PubMed Scopus (280) Google Scholar, 10Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Additionally, these secondary structures may also prevent the access of repair enzymes to damaged telomeric DNA. Thus, telomeric DNA presents a challenge to DNA replication and repair, and an appropriate coordination between specialized DNA replication/repair proteins and the shelterin proteins is essential for telomere integrity. The RecQ helicases WRN and BLM are known to be involved in telomere maintenance (7Verdun R.E. Karlseder J. Nature. 2007; 447: 924-931Crossref PubMed Scopus (379) Google Scholar, 11Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kølvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 12Lillard-Wetherell K. Machwe A. Langland G.T. Combs K.A. Behbehani G.K. Schonberg S.A. German J. Turchi J.J. Orren D.K. Groden J. Hum. Mol. Genet. 2004; 13: 1919-1932Crossref PubMed Scopus (124) Google Scholar, 13Opresko P.L. Mason P.A. Podell E.R. Lei M. Hickson I.D. Cech T.R. Bohr V.A. J. Biol. Chem. 2005; 280: 32069-32080Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The shelterin protein TRF1 interacts with WRN in vitro and modulates its activity at the telomere (11Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kølvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). It has also been suggested that TRF1 may recruit BLM, which can resolve G-quadruplex structures efficiently (10Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Likewise, another shelterin protein, TRF2, interacts with both WRN and BLM and stimulates their helicase activities on telomeric D-loops in vitro, suggesting that TRF2 may play a role in the recruitment of these proteins at telomeres (11Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kølvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). RecQ helicases are a highly conserved family of proteins that play significant roles in DNA metabolic processes including DNA replication, DNA repair, and DNA recombination. Saccharomyces cerevisiae and Schizosaccharomyces pombe each express only a single RecQ helicase (Sgs1 and Rqh1, respectively), whereas five RecQ homologs are expressed in mammalian cells: RECQ1, BLM, WRN, RECQL4, and RECQ5 (14Bohr V.A. Trends Biochem. Sci. 2008; 33: 609-620Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 15Chu W.K. Hickson I.D. Nat. Rev. Cancer. 2009; 9: 644-654Crossref PubMed Scopus (361) Google Scholar). BLM, WRN, and RECQL4 are linked to autosomal recessive disorders characterized by genomic instability and cancer predisposition. Bloom syndrome and Werner syndrome are associated with defects in BLM and WRN, respectively (16Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (578) Google Scholar, 17Yu 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: 258-262Crossref PubMed Scopus (1488) Google Scholar), whereas RECQL4 deficiency is associated with three rare autosomal recessive diseases: Rothmund-Thomson syndrome (RTS), Baller-Gerold syndrome, and RAPADILINO syndrome (18Larizza L. Roversi G. Volpi L. Orphanet J. Rare Dis. 2010; 5: 2Crossref PubMed Scopus (189) Google Scholar, 19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar). BLM and WRN play important roles in DNA repair and replication (14Bohr V.A. Trends Biochem. Sci. 2008; 33: 609-620Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 20Popuri V. Bachrati C.Z. Muzzolini L. Mosedale G. Costantini S. Giacomini E. Hickson I.D. Vindigni A. J. Biol. Chem. 2008; 283: 17766-17776Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 21Brosh Jr., R.M. Karow J.K. White E.J. Shaw N.D. Hickson I.D. Bohr V.A. Nucleic Acids Res. 2000; 28: 2420-2430Crossref PubMed Scopus (56) Google Scholar, 22Walpita D. Plug A.W. Neff N.F. German J. Ashley T. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 5622-5627Crossref PubMed Scopus (82) Google Scholar) and have also been implicated in telomere maintenance. Although the biological function of RECQL4 is not well established, it has been proposed that it participates in base excision repair, nucleotide excision repair, and homologous recombination (23Singh D.K. Karmakar P. Aamann M. Schurman S.H. May A. Croteau D.L. Burks L. Plon S.E. Bohr V.A. Aging Cell. 2010; 9: 358-371Crossref PubMed Scopus (72) Google Scholar, 24Jin W. Liu H. Zhang Y. Otta S.K. Plon S.E. Wang L.L. Hum. Genet. 2008; 123: 643-653Crossref PubMed Scopus (61) Google Scholar, 25Liu Y. DNA Repair. 2010; 9: 325-330Crossref PubMed Scopus (32) Google Scholar, 26Schurman S.H. Hedayati M. Wang Z. Singh D.K. Speina E. Zhang Y. Becker K. Macris M. Sung P. Wilson 3rd, D.M. Croteau D.L. Bohr V.A. Hum. Mol. Genet. 2009; 18: 3470-3483Crossref PubMed Scopus (71) Google Scholar). In addition, some studies suggest that Xenopus laevis RECQL4 is active in the initiation of DNA replication (27Matsuno K. Kumano M. Kubota Y. Hashimoto Y. Takisawa H. Mol. Cell. Biol. 2006; 26: 4843-4852Crossref PubMed Scopus (140) Google Scholar, 28Sangrithi M.N. Bernal J.A. Madine M. Philpott A. Lee J. Dunphy W.G. Venkitaraman A.R. Cell. 2005; 121: 887-898Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Consistent with this, human RECQL4 interacts with the minichromosome maintenance complex (29Xu X. Rochette P.J. Feyissa E.A. Su T.V. Liu Y. EMBO J. 2009; 28: 3005-3014Crossref PubMed Scopus (106) Google Scholar) and the origin of replication (30Thangavel S. Mendoza-Maldonado R. Tissino E. Sidorova J.M. Yin J. Wang W. Monnat Jr., R.J. Falaschi A. Vindigni A. Mol. Cell. Biol. 2010; 30: 1382-1396Crossref PubMed Scopus (107) Google Scholar) during replication initiation. RTS patients who do not die from cancer have a normal life span. However, they show features of “segmental premature aging” such as growth retardation, poikiloderma, hair loss, cataracts, and bony malformations, and thus RTS is considered a premature aging syndrome (31Hoki Y. Araki R. Fujimori A. Ohhata T. Koseki H. Fukumura R. Nakamura M. Takahashi H. Noda Y. Kito S. Abe M. Hum. Mol. Genet. 2003; 12: 2293-2299Crossref PubMed Scopus (91) Google Scholar, 32Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Crossref PubMed Scopus (190) Google Scholar). Interestingly, some RTS patients have phenotypes similar to dyskeratosis congenita, which is caused mainly by telomere abnormalities (33Walne A.J. Vulliamy T. Beswick R. Kirwan M. Dokal I. Hum. Mol. Genet. 2010; 19: 4453-4461Crossref PubMed Scopus (79) Google Scholar). Further, RTS and Werner syndrome share many clinical features, including developmental abnormalities, premature aging, and a high degree of susceptibility to osteosarcomas (34Stinco G. Governatori G. Mattighello P. Patrone P. J. Dermatol. 2008; 35: 154-161Crossref PubMed Scopus (47) Google Scholar, 35Vennos E.M. Collins M. James W.D. J. Am. Acad. Dermatol. 1992; 27: 750-762Abstract Full Text PDF PubMed Scopus (146) Google Scholar). WRN interacts with telomeric structures and plays a significant role in telomere replication and repair (11Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kølvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 36Ghosh A. Rossi M.L. Aulds J. Croteau D. Bohr V.A. J. Biol. Chem. 2009; 284: 31074-31084Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Recent studies show that RECQL4 has a previously undetected helicase activity with selective DNA substrate specificity (19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar, 37Xu X. Liu Y. EMBO J. 2009; 28: 568-577Crossref PubMed Scopus (96) Google Scholar). RECQL4 also interacts with FEN1 (26Schurman S.H. Hedayati M. Wang Z. Singh D.K. Speina E. Zhang Y. Becker K. Macris M. Sung P. Wilson 3rd, D.M. Croteau D.L. Bohr V.A. Hum. Mol. Genet. 2009; 18: 3470-3483Crossref PubMed Scopus (71) Google Scholar), which may also play a role in telomere maintenance (38Saharia A. Teasley D.C. Duxin J.P. Dao B. Chiappinelli K.B. Stewart S.A. J. Biol. Chem. 2010; 285: 27057-27066Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Together, these observations suggest that RECQL4, like WRN, may play a role in telomere maintenance. We set out to look for telomeric abnormalities in RTS patient cells and RECQL4-depleted human cells and characterized interactions between RECQL4, shelterin proteins, telomeric D-loops, and WRN. The results showed that RECQL4 localizes at telomeres in replicative human cells and that the frequency of fragile telomeres is higher in RECQL4-depleted cells than in control cells, especially after DNA replication stress induced by aphidicolin exposure. Although RECQL4 can barely resolve telomeric D-loops, this activity is considerably enhanced in the presence of TRF1 or TRF2. RECQL4 also associates with WRN and stimulates WRN unwinding of telomeric D-loops in vitro. Collectively, our data support the view that RECQL4 participates in telomere maintenance by assisting in D-loop resolution. Wild-type (WT) human RECQL4 and helicase-dead RECQL4 with a C-terminal His9 tag in the pGEX6p1 vector (GE Healthcare) was expressed and purified from Escherichia coli Rosetta2 (DE3) (Novagen) as described previously (19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar). Recombinant histidine-tagged BLM was overexpressed in S. cerevisiae and purified as described previously (39Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Recombinant histidine-tagged wild-type WRN protein, recombinant GST-tagged human POT1 protein, and recombinant histidine-tagged human TRF2 and TRF1 protein were purified using a baculovirus/insect cell expression as described previously (4Lei M. Podell E.R. Cech T.R. Nat. Struct. Mol. Biol. 2004; 11: 1223-1229Crossref PubMed Scopus (365) Google Scholar, 11Opresko P.L. Otterlei M. Graakjaer J. Bruheim P. Dawut L. Kølvraa S. May A. Seidman M.M. Bohr V.A. Mol. Cell. 2004; 14: 763-774Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 40Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar). Protein concentration was determined by the Bradford assay (Bio-Rad), and purity was determined by SDS-PAGE and Coomassie staining. All of the unmodified oligonucleotides were from Integrated DNA Technologies (Coralville, IA) and were PAGE-purified by the manufacturer. The modified (8-oxo-2-deoxyguanosine-containing) oligonucleotides were synthesized and purified by The Midland Certified Reagent Co. (Midland, TX). The D-loops were prepared and characterized as described previously (36Ghosh A. Rossi M.L. Aulds J. Croteau D. Bohr V.A. J. Biol. Chem. 2009; 284: 31074-31084Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). All assays were performed at least in triplicate. RECQL4 helicase assays were performed as described earlier (19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar). POT1, TRF1, and TRF2 (amounts indicated in the legends of FIGURE 7, FIGURE 8, FIGURE 9) were added together with RECQL4 when indicated. WRN and BLM helicase reactions were performed and analyzed similarly, only in a different reaction buffer (40 mm Tris, pH 8.0, 4 mm MgCl2, 5 mm DTT, 2 mm ATP, and 100 μg/ml BSA). While investigating the effect of RECQL4 on WRN and BLM helicase activities, the indicated amounts of RECQL4 were added together with WRN or BLM. DNA substrate and protein concentrations were as indicated in the legends of FIGURE 7, FIGURE 8, FIGURE 9.FIGURE 8RECQL4 physically and functionally interacts with TRF2. A, autoradiogram showing the unwinding activity of RECQL4 (10 nm) on 0.5 nm DL1 in the presence of 0, 5, 10, and 20 nm TRF1 (lanes 1–5) and TRF2 (lanes 6–9) in the presence of 25× excess single-stranded DNA. Δ (lane 12), represents heat-denatured DL1. 20 nm TRF1 (lane 10) or TRF2 (lane 11) alone does not have any unwinding activity on DL1. B, histogram showing the unwinding activity of RECQL4 (10 nm) on 0.5 nm DL1 in the presence of 0, 5, 10, and 20 nm TRF1 and TRF2 in the presence of 25× excess single-stranded DNA. The error bars represent mean ± S.D., n = 3. C, autoradiogram showing the effect of 5, 10, and 20 nm TRF1 (lanes 1–5) or TRF2 (lanes 6–10) on annealing activity of RECQL4. 0.5 nm radiolabeled oligo SS1 and 2.5× excess oligo BB were used as annealing substrates. D, in vitro pulldown of TRF2 by RECQL4. Lane 1, represents input, showing the bands corresponding to RECQL4 and TRF2. Lane 2, represents co-IP with IgG controls. Lane 3, represents co-IP with antibodies specific to RECQL4. Bands corresponding to anti-RECQL4 and anti-TRF2 antibodies are marked with arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 9RECQL4 and WRN synergistically unwind telomeric D-loops. A, gel showing the effect of 10 and 5 nm RECQL4 on the unwinding activities of 10 nm WRN and 5 nm BLM, respectively. 0.5 nm DL1 (lanes 1–5) and DL4 (lanes 7–11) were used as substrates. Lanes 6 and 12, represent the unwinding activity of RECQL4 alone on DL1 and DL4, respectively. Δ (lane 13), indicates heat-denatured DL4. B, quantitative analysis of the gel showing the effect of RECQL4 on WRN and BLM unwinding activities on DL1 and DL4 at a 1:1 molar ratio of the corresponding proteins. The error bars represent mean ± S.D., n = 3. C, in vivo co-IP of WRN by FLAG-tagged RECQL4 in U2OS cells. The bands corresponding to anti-WRN and anti-FLAG antibodies are shown. Lanes 1 and 2, the bands in RECQL4-FLAG and empty vector-transduced cells, respectively. The corresponding IPs with FLAG, in the presence of EtBr, are shown in lanes 3 and 4, respectively. D, schematic summarizing the proposed synergistic role of RECQL4 and WRN in telomere maintenance. The DNA replication and repair machinery fails to proceed through the telomeric D-loop, and WRN and RECQL4 are recruited by the shelterin complex (most probably by TRF2, as both of these RecQ helicases interact with it directly) to resolve this structure and thus maintain telomere integrity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) RECQL4 (7.5, 15, and 30 nm) was incubated with either 0.5 nm non-telomeric D-loops (DLmx) or 0.5 nm telomeric D-loops (DL1) in a 10-μl reaction buffer containing 30 mm Tris-HCl, pH 7.4, 1 mm DTT, 100 μg/ml BSA, and 50 mm KCl for 15 min on ice. Then 5 μl of stop dye (50% glycerol and 0.05% bromphenol blue) was added. Reactions were kept on ice until loading onto a 1% agarose/20 mm Tris, 10 mm acetic acid, and 0.5 mm EDTA (0.5 × TAE) gel and run in 0.5× TAE at 200 V for 1.5 h at 4 °C. Gel was dried in a gel dryer and exposed to a PhosphorImager screen. The image was scanned and analyzed using ImageQuant TL (GE Healthcare). Assays were performed at least in triplicate, and a representative gel shown. U2OS and HeLa cells were maintained in high glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Invitrogen) at 37 °C in 5% CO2. Normal (GM00323 and GM01864) and RTS patient cell lines (AG05013 and AG18371) were obtained from Coriell Cell Repository (Camden, NJ). The cells were grown in Amniomax II medium (Invitrogen) and maintained at 37 °C in 5% CO2. Primary BJ cells were grown in Eagle's minimum essential medium (ATCC, Manassas, VA) with 10% fetal bovine serum (Sigma). RECQL4 knockdown (RECQL4 KD) and scrambled control cells (SCR) were prepared in primary BJ, U2OS, and HeLa cells according to the standard protocol using Mission shRNA lentiviral construct TRCN0000051169 from Sigma-Aldrich. Briefly, the shRNA vector was co-transfected with packing plasmid pCMV-dr8.2 DVPR (Addgene, Cambridge, MA) and envelope vector pCMV-VSV-G (Addgene) into human embryonic kidney 293T cells in DMEM with Hyclone FBS (Thermo Fisher Scientific) using FuGENE® 6 transfection reagent (Roche Applied Science) and Opti-MEM (Invitrogen). The medium was collected 48 h after transduction and filtered using a 0.45-μm PVDF membrane filter (Millipore). Lentivirus was applied to the cells, and infections were allowed to proceed for 48 h after which time puromycin was applied to select for those cells that had taken up the lentivirus. Quantitative PCR was performed to confirm that the RECQL4 was knocked down ∼90%, which was later confirmed by Western blotting prior to the cells being used in experiments. 100,000 U2OS cells were grown overnight in 6-well plates in 2 ml of antibiotic-free medium (DMEM plus 10% FBS). Then the cells were treated with 100 pmol of either control (Silencer Negative Control #1, Ambion) or RECQL4-targeted siRNA (target sequence CAAUACAGCUUACCGUACA, Dharmacon) in Lipofectamine 2000 reagent (Invitrogen) for 6 h. Cells were then grown overnight and again treated with same siRNAs for 6 h. Cells were then grown in antibiotic (1%)-containing medium and harvested after 72 h, and the RECQL4 level was checked by Western blotting. For in vitro co-IP, purified WRN (7.5 pmol) and purified RECQL4 (15 pmol) were mixed in a buffer containing 20 mm Tris-HCl, pH 7.4, 20 mm NaCl, 25 mm KCl, 1 mm DTT, 5% glycerol, 2.5 mm MgCl2 and 100 μg/ml BSA and incubated for 90 min at 4 °C. For RECQL4-TRF2 co-IP, the proteins were mixed in a buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and 1× protease inhibitors (Roche Applied Science). Then, precoated antibody-agarose beads (anti-rabbit WRN and normal rabbit IgG, Santa Cruz Biotechnology) were added to the protein mix, and the tubes were incubated for 2 h at 4 °C with end-over-end rotation. After incubation the beads were washed four times in washing buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, and 1% Triton X-100, resuspended in 30 μl of 2× SDS loading buffer, and resolved on a 4–12% gradient SDS-PAGE followed by Western analysis. The RECQL4 was detected by polyclonal anti rabbit-RECQL4 (19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar) and rabbit TrueBlot HRP-conjugated anti-rabbit IgG secondary antibody (eBioscience) using an ECL kit following the manufacturer's protocol (GE Healthcare). For in vivo co-IP of TRF2 by RECQL4, U2OS cells were lysed in cell lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and 1× protease inhibitors (Roche Applied Science)), and co-IP was performed as described elsewhere using anti-TRF2 (Imgenex, mouse) and anti-RECQL4 (19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar) (rabbit). For co-IP of WRN with FLAG-RECQL4, U2OS cells were first transduced with FLAG-RECQL4 and empty vector plasmids using Lipofectamine LTX following the manufacturer's protocol (Invitrogen). At 24 h post-transfection, the cells were lysed in 1 ml of cell lysis buffer in the presence of 50 μg/ml ethidium bromide. WRN was detected with polyclonal anti-rabbit WRN antibody (Santa Cruz Biotechnology), and FLAG-RECQL4 was detected with anti-rabbit FLAG antibody (Sigma) using ECL Plus (GE Healthcare). U2OS or HeLa cells (untreated, RECQL4-specific, or scrambled shRNA-treated, as indicated) were plated on Lab-Tek II chambered glass slides (Thermo-Fisher Scientific) at a density of 20,000 cells/chamber, grown overnight, and then treated with 2 gray IR where indicated. For cell cycle-dependent studies, cells were treated with 2 mm hydroxyurea (Sigma) or 2 mm nocodazole (Sigma) for 16 h as indicated. Hydroxyurea- and nocodazole-containing medium was then replaced by DMEM, and the cells were fixed immediately. Cells were fixed with 4% paraformaldehyde in PBS for 10 min at 37 °C, washed with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. Following a final wash with PBS, cells were blocked overnight with 5% FBS in PBS. Then the cells were treated with primary antibodies for 1 h at 37 °C and secondary antibodies for 30 min at 37 °C after washing in PBS (five times for 3 min). Then the cells were washed again with PBS (five times for 3 min) and treated with Vectashield mounting medium containing DAPI (Vector Laboratories). The following antibodies were used as described under “Results.” Primary antibodies were: 53BP1 (Novus, 1:100, rabbit), TRF1 (Abcam TRF-78, 1:50, mouse), TRF2 (Imgenex, 1:50, mouse), RECQL4 (in-house) (19Rossi M.L. Ghosh A.K. Kulikowicz T. Croteau D.L. Bohr V.A. DNA Repair. 2010; 9: 796-804Crossref PubMed Scopus (60) Google Scholar) (1:100, rabbit or Santa Cruz K-16, 1:100, goat), and WRN (Santa Cruz, 1:100, rabbit). Secondary antibodies were: donkey anti-rabbit Alexa Fluor 488 (1:1000) and donkey anti-mouse Alexa Fluor 647 (1:200 for TRF-78 and 1:1000 for others) (all from Invitrogen). All of the dilutions were prepared in blocking solution (5% FBS). Images were captured on a Nikon Eclipse TE2000 confocal microscope (×40 magnification) with a Hamamatsu C9100-13 camera at −65 °C. Data acquisition and analysis were performed using Volocity 5.5 software (PerkinElmer Life Sciences). Images of individual representative cells were cropped from the original images and are shown in Figs. 2, 3, and 5. To further clarify the coexistence of two signals, images of partially colocalized foci were cropped and visualized as three-dimensional images as shown in Figs. 2, 3, and 5. The Pearson coefficients of these individual foci were calculated using Volocity software. For a better representation (supplemental Fig. 3), colocalization channels (positive products of the differences of the means for the two channels) were generated to highlight the colocalized foci. For quantification purpose, the foci at which two signals are intersected (colocalized foci) are marked and calculated using the same software.FIGURE 3Telomeric 53BP1 foci in RECQL4 knockdown HeLa and U2OS cells. A, Western blot showing the level of RECQL4 in scrambled shRNA-treated (Scramble) and RECQL4-targeted shRNA-treated (RECQL4 KD) HeLa cells. Bands corresponding to RECQL4 and actin are shown by arrows. B, confocal microscopic images showing colocalization of 53BP1 foci (green) and TRF1 (red) signals in scrambled (panels 1–3) and RECQL4 KD HeLa cells (panels 4–6). Nuclear staining with DAPI is shown in the merged image. Some of the colocalized foci are marked with white arrows. Close-up images of some of the colocalized foci are shown next to panel 6. Scale bar = 5 μm. C, histograms showing the frequency distribution of telomeric 53BP1 foci (TIF) in scrambled and RECQL4 KD HeLa cells (n = 70). D, Western blot showing the level of RECQL4 in control siRNA-treated (Control) and RECQL4-targeted siRNA-treated (RECQL4 KD) U2OS cells. Bands corresponding to RECQL4 and actin are shown by arrows. E, confocal microscopic images showing colocalization of 53BP1 foci (green) and TRF1 (red) signals in control (panels 1–3) and RECQL4 KD U2OS cells (panels 4–6). Nuclear staining with DAPI is shown in the merged image. Some of the col" @default.
• W1997963513 created "2016-06-24" @default.
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• W1997963513 date "2012-01-01" @default.
• W1997963513 modified "2023-10-14" @default.
• W1997963513 title "RECQL4, the Protein Mutated in Rothmund-Thomson Syndrome, Functions in Telomere Maintenance" @default.
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• W1997963513 doi "https://doi.org/10.1074/jbc.m111.295063" @default.
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