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• W2034502973 abstract "The list of factors that participate in the DNA damage response to maintain genomic stability has expanded significantly to include a role for proteins involved in RNA processing. Here, we provide evidence that the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS) is a novel component of the DNA damage response. We demonstrate that FUS is rapidly recruited to sites of laser-induced DNA double-strand breaks (DSBs) in a manner that requires poly(ADP-ribose) (PAR) polymerase activity, but is independent of ataxia-telangiectasia mutated kinase function. FUS recruitment is mediated by the arginine/glycine-rich domains, which interact directly with PAR. In addition, we identify a role for the prion-like domain in promoting accumulation of FUS at sites of DNA damage. Finally, depletion of FUS diminished DSB repair through both homologous recombination and nonhomologous end-joining, implicating FUS as an upstream participant in both pathways. These results identify FUS as a new factor in the immediate response to DSBs that functions downstream of PAR polymerase to preserve genomic integrity.Background: FUS has been implicated in the DNA damage response; however, the mechanisms are unknown.Results: FUS recruitment to DNA lesions is PARP-dependent. Depletion of FUS disrupts DNA repair.Conclusion: FUS functions downstream of PARP and promotes double-strand break repair.Significance: This work identifies FUS as a novel factor at DNA lesions and furthers our understanding of RNA-binding proteins in maintaining genomic stability. The list of factors that participate in the DNA damage response to maintain genomic stability has expanded significantly to include a role for proteins involved in RNA processing. Here, we provide evidence that the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS) is a novel component of the DNA damage response. We demonstrate that FUS is rapidly recruited to sites of laser-induced DNA double-strand breaks (DSBs) in a manner that requires poly(ADP-ribose) (PAR) polymerase activity, but is independent of ataxia-telangiectasia mutated kinase function. FUS recruitment is mediated by the arginine/glycine-rich domains, which interact directly with PAR. In addition, we identify a role for the prion-like domain in promoting accumulation of FUS at sites of DNA damage. Finally, depletion of FUS diminished DSB repair through both homologous recombination and nonhomologous end-joining, implicating FUS as an upstream participant in both pathways. These results identify FUS as a new factor in the immediate response to DSBs that functions downstream of PAR polymerase to preserve genomic integrity. Background: FUS has been implicated in the DNA damage response; however, the mechanisms are unknown. Results: FUS recruitment to DNA lesions is PARP-dependent. Depletion of FUS disrupts DNA repair. Conclusion: FUS functions downstream of PARP and promotes double-strand break repair. Significance: This work identifies FUS as a novel factor at DNA lesions and furthers our understanding of RNA-binding proteins in maintaining genomic stability. Exposure to genotoxic agents including ionizing radiation (IR), 2The abbreviations used are: IRionizing radiationALSamyotrophic lateral sclerosisATMataxia-telangiectasia mutatedDDRDNA damage responseDSBdouble-strand breakFUSfused in sarcomaHRhomologous recombinationLIDDlaser-induced DNA damageNHEJnonhomologous end-joiningNTnontargetingPARpoly(ADP-ribose)PARPPAR polymerasePLDprion-like domainRRGarginine/glycine-richRRMRNA recognition motifSSBsingle-strand breakTDPTAR DNA-binding proteinZNFzinc finger. H2O2, and radiomimetic drugs poses a significant challenge to genomic integrity that is combated by evolutionarily conserved pathways that are collectively referred to as the DNA damage response (DDR) (1.Harper J.W. Elledge S.J. The DNA damage response: ten years after.Mol. Cell. 2007; 28: 739-745Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 2.Jackson S.P. Bartek J. The DNA-damage response in human biology and disease.Nature. 2009; 461: 1071-1078Crossref PubMed Scopus (3828) Google Scholar). Central to this paradigm is the activation of apical phosphoinositide 3-kinase-like kinases, including ATM, ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) that serve as proximal DNA damage signal transducers. The best characterized of these, ATM, is activated by double-strand breaks (DSBs) to phosphorylate an estimated 700 substrates (3.Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. Shiloh Y. Gygi S.P. Elledge S.J. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2356) Google Scholar) that impact cell cycle regulation, apoptosis, DNA repair, and many other cellular processes. ATM rapidly accumulates at sites of DSBs in an MRE11-RAD50-NBS1-dependent manner and phosphorylates the histone variant H2AX on serine 139 (γH2AX) (4.Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4141) Google Scholar, 5.Lee J.H. Paull T.T. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks.Oncogene. 2007; 26: 7741-7748Crossref PubMed Scopus (415) Google Scholar). γH2AX serves as a scaffold for the recruitment of the mediator protein MDC1 (6.Lou Z. Minter-Dykhouse K. Franco S. Gostissa M. Rivera M.A. Celeste A. 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Chem. 2008; 283: 13549-13555Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 10.Mailand N. Bekker-Jensen S. Faustrup H. Melander F. Bartek J. Lukas C. Lukas J. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins.Cell. 2007; 131: 887-900Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 11.Wang B. Elledge S.J. Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20759-20763Crossref PubMed Scopus (365) Google Scholar, 12.Stewart G.S. Panier S. Townsend K. Al-Hakim A.K. Kolas N.K. Miller E.S. Nakada S. Ylanko J. Olivarius S. Mendez M. Oldreive C. Wildenhain J. Tagliaferro A. Pelletier L. Taubenheim N. Durandy A. Byrd P.J. Stankovic T. Taylor A.M. Durocher D. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.Cell. 2009; 136: 420-434Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 13.Doil C. Mailand N. Bekker-Jensen S. Menard P. Larsen D.H. Pepperkok R. Ellenberg J. Panier S. Durocher D. Bartek J. Lukas J. Lukas C. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins.Cell. 2009; 136: 435-446Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). RNF8 and RNF168 activities are required for the recruitment of the RAP80-ABRA1-BRCA1 complex (12.Stewart G.S. Panier S. Townsend K. Al-Hakim A.K. Kolas N.K. Miller E.S. Nakada S. Ylanko J. Olivarius S. Mendez M. Oldreive C. Wildenhain J. Tagliaferro A. Pelletier L. Taubenheim N. Durandy A. Byrd P.J. Stankovic T. Taylor A.M. Durocher D. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.Cell. 2009; 136: 420-434Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 13.Doil C. Mailand N. Bekker-Jensen S. Menard P. Larsen D.H. Pepperkok R. Ellenberg J. Panier S. Durocher D. Bartek J. Lukas J. Lukas C. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins.Cell. 2009; 136: 435-446Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar) and p53-binding protein 1 (53BP1) (10.Mailand N. Bekker-Jensen S. Faustrup H. Melander F. Bartek J. Lukas C. Lukas J. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins.Cell. 2007; 131: 887-900Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 12.Stewart G.S. Panier S. Townsend K. Al-Hakim A.K. Kolas N.K. Miller E.S. Nakada S. Ylanko J. Olivarius S. Mendez M. Oldreive C. Wildenhain J. Tagliaferro A. Pelletier L. Taubenheim N. Durandy A. Byrd P.J. Stankovic T. Taylor A.M. Durocher D. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.Cell. 2009; 136: 420-434Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 13.Doil C. Mailand N. Bekker-Jensen S. Menard P. Larsen D.H. Pepperkok R. Ellenberg J. Panier S. Durocher D. Bartek J. Lukas J. Lukas C. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins.Cell. 2009; 136: 435-446Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 14.Kolas N.K. Chapman J.R. Nakada S. Ylanko J. Chahwan R. Sweeney F.D. Panier S. Mendez M. Wildenhain J. Thomson T.M. Pelletier L. Jackson S.P. Durocher D. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.Science. 2007; 318: 1637-1640Crossref PubMed Scopus (706) Google Scholar). The composition of the recruitment complexes may dictate whether DSBs are repaired via NHEJ or HR repair mechanisms (15.Chapman J.R. Taylor M.R. Boulton S.J. Playing the end game: DNA double-strand break repair pathway choice.Mol. Cell. 2012; 47: 497-510Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar). Working in parallel to the γH2AX-mediated recruitment pathway is a PARP-dependent pathway that responds primarily to DNA single-strand breaks (SSBs) and participates in SSB repair and alternative-NHEJ (16.Langelier M.F. Planck J.L. Roy S. Pascal J.M. Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1.Science. 2012; 336: 728-732Crossref PubMed Scopus (413) Google Scholar, 17.Eustermann S. Videler H. Yang J.C. Cole P.T. Gruszka D. Veprintsev D. Neuhaus D. The DNA-binding domain of human PARP-1 interacts with DNA single-strand breaks as a monomer through its second zinc finger.J. Mol. Biol. 2011; 407: 149-170Crossref PubMed Scopus (115) Google Scholar, 18.Huber A. Bai P. de Murcia J.M. de Murcia G. PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development.DNA Repair. 2004; 3: 1103-1108Crossref PubMed Scopus (189) Google Scholar, 19.Wang M. Wu W. Wu W. Rosidi B. Zhang L. Wang H. Iliakis G. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.Nucleic Acids Res. 2006; 34: 6170-6182Crossref PubMed Scopus (594) Google Scholar). PARP ADP-ribosylates target proteins including histones and itself at sites of damage, which creates binding sites for proteins harboring PAR binding domains (20.De Vos M. Schreiber V. Dantzer F. The diverse roles and clinical relevance of PARPs in DNA damage repair: current state of the art.Biochem. Pharmacol. 2012; 84: 137-146Crossref PubMed Scopus (372) Google Scholar). PARP is required for the recruitment of CHD4-NuRD and Polycomb group transcriptional repressor complexes, which mediate histone deacetylation and chromatin compaction near the break site, presumably to reduce interference between transcription and DSB repair (21.Polo S.E. Kaidi A. Baskcomb L. Galanty Y. Jackson S.P. Regulation of DNA-damage responses and cell cycle progression by the chromatin remodelling factor CHD4.EMBO J. 2010; 29: 3130-3139Crossref PubMed Scopus (258) Google Scholar, 22.Chou D.M. Adamson B. Dephoure N.E. Tan X. Nottke A.C. Hurov K.E. Gygi S.P. Colaiácovo M.P. Elledge S.J. A chromatin localization screen reveals poly(ADP ribose)-regulated recruitment of the repressive Polycomb and NuRD complexes to sites of DNA damage.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 18475-18480Crossref PubMed Scopus (415) Google Scholar, 23.Smeenk G. Wiegant W.W. Vrolijk H. Solari A.P. Pastink A. van Attikum H. The NuRD chromatin-remodeling complex regulates signaling and repair of DNA damage.J. Cell Biol. 2010; 190: 741-749Crossref PubMed Scopus (180) Google Scholar, 24.Lai A.Y. Wade P.A. Cancer biology and NuRD: a multifaceted chromatin remodelling complex.Nat. Rev. Cancer. 2011; 11: 588-596Crossref PubMed Scopus (346) Google Scholar). ionizing radiation amyotrophic lateral sclerosis ataxia-telangiectasia mutated DNA damage response double-strand break fused in sarcoma homologous recombination laser-induced DNA damage nonhomologous end-joining nontargeting poly(ADP-ribose) PAR polymerase prion-like domain arginine/glycine-rich RNA recognition motif single-strand break TAR DNA-binding protein zinc finger. FUS is a 526-amino acid member of the FET family of RBPs, which include Ewing sarcoma (EWSR1), Tata-binding protein-associated factor 2N (TAF15), and the Drosophila ortholog of FUS, SARFH/Cabeza (25.Bertolotti A. Lutz Y. Heard D.J. Chambon P. Tora L. hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II.EMBO J. 1996; 15: 5022-5031Crossref PubMed Scopus (310) Google Scholar, 26.Stolow D.T. Haynes S.R. Cabeza, a Drosophila gene encoding a novel RNA-binding protein, shares homology with EWS and TLS, two genes involved in human sarcoma formation.Nucleic Acids Res. 1995; 23: 835-843Crossref PubMed Scopus (67) Google Scholar). FUS was initially identified as a fusion oncogene in myxoid liposarcoma, in which the transcriptional activation domain of FUS is fused to the C/EBP homologous protein (CHOP) (27.Crozat A. Aman P. Mandahl N. Ron D. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma.Nature. 1993; 363: 640-644Crossref PubMed Scopus (750) Google Scholar, 28.Rabbitts T.H. Forster A. Larson R. Nathan P. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma.Nat. Genet. 1993; 4: 175-180Crossref PubMed Scopus (476) Google Scholar). In addition, FUS fusion proteins have been identified in a variety of human cancers including acute myeloid leukemia, angiomatoid fibrous histiocytoma, and fibromyxoid sarcoma (29.Law W.J. Cann K.L. Hicks G.G. TLS, EWS and TAF15: a model for transcriptional integration of gene expression.Brief Funct. Genomic Proteomic. 2006; 5: 8-14Crossref PubMed Scopus (156) Google Scholar). FUS is composed of N-terminal Gln/Gly/Ser/Tyr-rich and Gly-rich regions that comprise a prion-like domain (PLD) (30.Gitler A.D. Shorter J. RNA-binding proteins with prion-like domains in ALS and FTLD-U.Prion. 2011; 5: 179-187Crossref PubMed Scopus (114) Google Scholar), an RNA recognition motif (RRM), arginine/glycine-rich (RGG) domains, and a C-terminal zinc finger domain (ZNF) (see Fig. 2A) (31.Iko Y. Kodama T.S. Kasai N. Oyama T. Morita E.H. Muto T. Okumura M. Fujii R. Takumi T. Tate S. Morikawa K. Domain architectures and characterization of an RNA-binding protein, TLS.J. Biol. Chem. 2004; 279: 44834-44840Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). FUS binds RNA (32.Prasad D.D. Ouchida M. Lee L. Rao V.N. Reddy E.S. TLS/FUS fusion domain of TLS/FUS-erg chimeric protein resulting from the t(16;21) chromosomal translocation in human myeloid leukemia functions as a transcriptional activation domain.Oncogene. 1994; 9: 3717-3729PubMed Google Scholar, 33.Zinszner H. Sok J. Immanuel D. Yin Y. Ron D. TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling.J. Cell Sci. 1997; 110: 1741-1750Crossref PubMed Google Scholar) and ssDNA and dsDNA (33.Zinszner H. Sok J. Immanuel D. Yin Y. Ron D. TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling.J. Cell Sci. 1997; 110: 1741-1750Crossref PubMed Google Scholar, 34.Baechtold H. Kuroda M. Sok J. Ron D. Lopez B.S. Akhmedov A.T. Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUS and is able to promote D-loop formation.J. Biol. Chem. 1999; 274: 34337-34342Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 35.Perrotti D. Bonatti S. Trotta R. Martinez R. Skorski T. Salomoni P. Grassilli E. Lozzo R.V. Cooper D.R. Calabretta B. TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.EMBO J. 1998; 17: 4442-4455Crossref PubMed Scopus (121) Google Scholar) and has been shown to shuttle between the nucleus and cytoplasm (33.Zinszner H. Sok J. Immanuel D. Yin Y. Ron D. TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling.J. Cell Sci. 1997; 110: 1741-1750Crossref PubMed Google Scholar). In addition to its participation in transcription, FUS has proposed physiological activities involving microRNA processing, splicing, and mRNA transport and maturation (36.Lagier-Tourenne C. Polymenidou M. Cleveland D.W. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration.Hum. Mol. Genet. 2010; 19: R46-R64Crossref PubMed Scopus (717) Google Scholar, 37.Schwartz J.C. Ebmeier C.C. Podell E.R. Heimiller J. Taatjes D.J. Cech T.R. FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser-2.Genes Dev. 2012; 26: 2690-2695Crossref PubMed Scopus (147) Google Scholar). Thus, FUS seems to fulfill broad functions in gene expression through transcriptional and posttranscriptional mechanisms. Intriguingly, dominant mutations in FUS cause inherited forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (38.Vance C. Rogelj B. Hortobágyi T. De Vos K.J. Nishimura A.L. Sreedharan J. Hu X. Smith B. Ruddy D. Wright P. Ganesalingam J. Williams K.L. Tripathi V. Al-Saraj S. Al-Chalabi A. Leigh P.N. Blair I.P. Nicholson G. de Belleroche J. Gallo J.M. Miller C.C. Shaw C.E. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6.Science. 2009; 323: 1208-1211Crossref PubMed Scopus (1931) Google Scholar, 39.Kwiatkowski Jr., T.J. Bosco D.A. Leclerc A.L. Tamrazian E. Vanderburg C.R. Russ C. Davis A. Gilchrist J. Kasarskis E.J. Munsat T. Valdmanis P. Rouleau G.A. Hosler B.A. Cortelli P. de Jong P.J. Yoshinaga Y. Haines J.L. Pericak-Vance M.A. Yan J. Ticozzi N. Siddique T. McKenna-Yasek D. Sapp P.C. Horvitz H.R. Landers J.E. Brown Jr., R.H. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis.Science. 2009; 323: 1205-1208Crossref PubMed Scopus (1956) Google Scholar). It is believed that ALS-associated mutations lead to trapping and aggregation of FUS in the cytoplasm; however, other pathogenic mechanisms may be at play. FUS also has an emergent, yet poorly understood participation in the DDR. FUS−/− mice exhibit defects in B lymphocyte development and activation, male sterility, chromosomal instability, and radiosensitivity, phenotypes that are closely aligned with DSB repair defects (40.Kuroda M. Sok J. Webb L. Baechtold H. Urano F. Yin Y. Chung P. de Rooij D.G. Akhmedov A. Ashley T. Ron D. Male sterility and enhanced radiation sensitivity in TLS−/− mice.EMBO J. 2000; 19: 453-462Crossref PubMed Scopus (179) Google Scholar, 41.Hicks G.G. Singh N. Nashabi A. Mai S. Bozek G. Klewes L. Arapovic D. White E.K. Koury M.J. Oltz E.M. Van Kaer L. Ruley H.E. Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death.Nat. Genet. 2000; 24: 175-179Crossref PubMed Scopus (230) Google Scholar). Cellular extracts from FUS−/− testes are unable to promote pairing between homologous DNA sequences in vitro (41.Hicks G.G. Singh N. Nashabi A. Mai S. Bozek G. Klewes L. Arapovic D. White E.K. Koury M.J. Oltz E.M. Van Kaer L. Ruley H.E. Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death.Nat. Genet. 2000; 24: 175-179Crossref PubMed Scopus (230) Google Scholar), and FUS was shown to promote D-loop formation (34.Baechtold H. Kuroda M. Sok J. Ron D. Lopez B.S. Akhmedov A.T. Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUS and is able to promote D-loop formation.J. Biol. Chem. 1999; 274: 34337-34342Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), an essential step in the repair of DSBs through the HR pathway (42.Thompson L.H. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.Mutat. Res. 2012; 751: 158-246Crossref PubMed Scopus (264) Google Scholar). Thus, the available evidence suggests that FUS participates in HR repair, possibly through direct actions at DSBs. FUS may also regulate the DDR through transcriptional mechanisms. Wang et al. showed that FUS is recruited to the cyclin D1 (CCND1) promoter through an interaction with sense and antisense noncoding CCND1 RNAs. Through inhibition of CREB-binding protein, FUS acts to repress CCND1 expression in response to DNA damage (43.Wang X. Arai S. Song X. Reichart D. Du K. Pascual G. Tempst P. Rosenfeld M.G. Glass C.K. Kurokawa R. Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription.Nature. 2008; 454: 126-130Crossref PubMed Scopus (838) Google Scholar). Finally, the finding that FUS is directly phosphorylated by ATM on Ser-42 and possibly neighboring phosphoinositide 3-kinase-like kinase consensus motifs in response to DNA damage provides strong circumstantial support for its role in the DDR (44.Gardiner M. Toth R. Vandermoere F. Morrice N.A. Rouse J. Identification and characterization of FUS/TLS as a new target of ATM.Biochem. J. 2008; 415: 297-307Crossref PubMed Scopus (76) Google Scholar); however, the functional significance of these posttranslational modifications is uncertain. Here, we demonstrate that FUS functions in cis to DNA damage via a PARP-dependent mechanism, which is mediated partially, if not completely, through binding of the RGG2 domain to PAR. In addition, we identify a role for the N-terminal PLD in accumulation of FUS at sites of DNA damage. Finally, FUS is required for efficient DNA repair through both HR and NHEJ pathways and for radioresistance. These data establish that FUS is dynamically regulated by DNA damage and functions downstream of PARP to maintain the stability of the genome. U-2 OS and HEK-293T cell lines were obtained from the American Type Culture Collection (ATCC). The HEK-293 cell lines EJ5-GFP and DR-GFP were a kind gift from Dr. Jeremy Stark (Beckman Research Institute of the City of Hope) (45.Pierce A.J. Johnson R.D. Thompson L.H. Jasin M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.Genes Dev. 1999; 13: 2633-2638Crossref PubMed Scopus (1048) Google Scholar, 46.Bennardo N. Cheng A. Huang N. Stark J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.PLoS Genet. 2008; 4: e1000110Crossref PubMed Scopus (608) Google Scholar). HEK-293T and HEK-293 cell lines were grown in Dulbecco's modified Eagle's medium (Cellgro). The U-2 OS cell line was grown in McCoy's medium. ATM (KU-55933) and PARP (PJ34) inhibitors (Calbiochem) were used at a final concentration of 20 μm and 1 μm, respectively. DNA-PK inhibitor (NU-7441) (R & D Systems) was used at a final concentration of 5 μm. All of the inhibitors were applied 1 h prior to subsequent analysis. IR was delivered using a JL Shepherd model JL-109 irradiator with a 137Cs source at 4.03 grays/min. The GFP-Myc-FUS(WT), GFP-Myc-FUS(R521G) and GFP-Myc-FUS(R524S) plasmids were a kind gift from Dr. Robert Baloh (Cedars-Sinai Medical Center). The GFP-FUS plasmid was a kind gift from Dr. Lawrence Hayward (University of Massachusetts Medical School) and was used for the generation of the following mutants: S42A, S26A/S42A/S61A/S84A (FUS4A), and C428A/C444A/C447A. pCI-NEO FUS(4F-L) was a kind gift from Dr. Udai Pandey (Louisiana State University Health Sciences Center) and was cloned into pCDNA5/FRT/TO/GFP (Addgene plasmid 19444 (47.Hageman J. Kampinga H.H. Computational analysis of the human HSPH/HSPA/DNAJ family and cloning of a human HSPH/HSPA/DNAJ expression library.Cell Stress Chaperones. 2009; 14: 1-21Crossref PubMed Scopus (123) Google Scholar). EWSR1 was cloned into pCDNA5/FRT/TO/GFP from pDEST/EWSR1 (Addgene plasmid 26377) (48.Hoell J.I. Larsson E. Runge S. Nusbaum J.D. Duggimpudi S. Farazi T.A. Hafner M. Borkhardt A. Sander C. Tuschl T. RNA targets of wild-type and mutant FET family proteins.Nat. Struct. Mol. Biol. 2011; 18: 1428-1431Crossref PubMed Scopus (249) Google Scholar). The mCherry and I-SceI plasmids were a kind gift from Dr. Sandra Weller (University of Connecticut Health Center). Point mutants were generated using primers designed by the QuikChange primer design program from Agilent Technologies. N-terminal truncation mutants (Δ1–285, Δ1–374, Δ1–467) were created using primers generating an internal start codon and were cloned into pCDNA5/FRT/TO/GFP. All primers were purchased from Integrated DNA Technologies (IDT). The internal deletion mutant, Δ204–475, was generated through the digestion of a full-length PCR product with BSPHI and EcoRI, which removes the RRM (33.Zinszner H. Sok J. Immanuel D. Yin Y. Ron D. TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling.J. Cell Sci. 1997; 110: 1741-1750Crossref PubMed Google Scholar). The fragments flanking the RRM were purified and ligated to yield Δ204–475. Mutations were verified by direct sequencing. All transfections were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Cells were analyzed 24–72 h after transfection. U-2 OS cells were plated onto 35-mm glass-bottom dishes (MatTek Corporation). Cells were either transfected using the outlined procedure or used directly for microirradiation. Cells were presensitized with 10 μg/ml Hoechst 33342 (Invitrogen) for 20 min at 37 °C. Microirradiation was performed with an A1R confocal microscope (Nikon) equipped with a 37 °C heating stage and 405-nm laser diode focused through a 60× Plan APO VC/1.4 oil objective. All microirradiation was performed using a laser power output of 40%. Live cell imaging was monitored using NIS-Elements viewer software (Nikon). Quantification of images was performed using ImageJ (National Institutes of Health) software. The pLKO.1 system was used to package lentiviruses and deliver short hairpin RNA (shRNA). The following shRNA target sequences were designed using the RNAi Consortium online tool (Broad Institute) and were cloned into pLKO.1-TRC (Addgene plasmid 10878) (49.Stewart S.A. Dykxhoorn D.M. Palliser D. Mizuno H. Yu E.Y. An D.S. Sabatini D.M. Chen I.S. Hahn W.C. Sharp P.A. Weinberg R.A. Novina C.D. Lentivirus-delivered stable gene silencing by RNAi in primary cells.RNA. 2003; 9: 493-501Crossref PubMed Scopus (1002) Google Scholar), according to the manufacturer's suggestions: FUS Coding Sequence, 5′-ATGAATGCAACCAGTGTAAGG-3′; FUS 3′-UTR, 5′-CAATTCCTGATCACCCAAGGG-3′; CtIP, 5′-CGGCAGCAGAATCTTAAACTT-3′; Lig4, 5′-GCCCGTGAATATGATTGCTAT-3′. Addgene plasmid 1864 containing a nontargeting (NT) shRNA was used as the control (50.Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar). Lentiviral particles were produced by transient transfection of HEK-293T cells with pLKO.1, psPAX2 (Addgene plasmid 12260), and pMD2.G (Addgene plasmid 12259) in a ratio of 4:3:1. U-2 OS, HEK-293 EJ5-GFP, and HEK-293 DR-GFP cells were infected with lentivirus and maintained in 1.5 μg/ml puromycin. Cells adhering to glass coverslips were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 in PBS at room temperature. Where indicated, cells were preextracted with CSK buffer (10 mm HEPES, pH 7.4, 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 0.5% Triton X-100) for 2 min on ice. Cells were blocked in 3% BSA for 30 min at room temperature and then incubated overnight at 4 °C with the indicated antibodies including: EWSR1 (Millipore DR1063), γH2AX (Millipore 05636), FUS (Santa Cruz Biotechnology 47711), and MDC1 (Sigma HPA006915). Cells were then incubated with either Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (Invitrogen) for 1 h at room temperature. Images were captured using an A1R confocal microscope equipped with a 60× Plan APO VC/1.4 oil objective and processed using Image J software. U-2 OS, HEK-293 EJ5-GFP, and HEK-293 DR-GFP cells were washed with PBS and treated in ice-cold cell lysis buffer (50 mm Tris, pH 7.5, 300 mm NaCl, 10% glycerol, 0.5% Triton X-100, 2 mm MgCl2, 3 mm EDTA) supplemented with protease and phosphatase inhibitors. Cell extracts were clarified by centrifugation at 20,000 × g for 10 min. Proteins were denatured and resolved by SDS-PAGE, transferred t" @default.
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• W2034502973 date "2013-08-01" @default.
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• W2034502973 title "The RNA-binding Protein Fused in Sarcoma (FUS) Functions Downstream of Poly(ADP-ribose) Polymerase (PARP) in Response to DNA Damage" @default.
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