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• W2970440112 abstract "•L1 ORF2p interacts with a diverse set of DNA replication and repair proteins•PARP2 is recruited to L1 ORF2p-induced single-strand DNA breaks•Activated (PARylated) PARP2 recruits RPA to L1 integration sites•PARP2 and RPA are required for efficient L1 retrotransposition Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome. Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome. An average individual genome contains ∼80–100 full-length long interspersed element-1 (LINE-1 or L1) sequences that can move to new locations (Brouha et al., 2003Brouha B. Schustak J. Badge R.M. Lutz-Prigge S. Farley A.H. Moran J.V. Kazazian Jr., H.H. Hot L1s account for the bulk of retrotransposition in the human population.Proc. Natl. Acad. Sci. USA. 2003; 100: 5280-5285Crossref PubMed Scopus (733) Google Scholar, Sassaman et al., 1997Sassaman D.M. Dombroski B.A. Moran J.V. Kimberland M.L. Naas T.P. DeBerardinis R.J. Gabriel A. Swergold G.D. Kazazian Jr., H.H. Many human L1 elements are capable of retrotransposition.Nat. Genet. 1997; 16: 37-43Crossref PubMed Scopus (364) Google Scholar) by a process termed retrotransposition. Full-length retrotransposition-competent human L1s (RC-L1s) are ∼6 kb and contain a 5′ UTR harboring sense and anti-sense RNA polymerase II promoters, two open reading frames (ORF1 and ORF2), and a 3′ UTR that ends in a poly(A) tract (Richardson et al., 2015Richardson S.R. Doucet A.J. Kopera H.C. Moldovan J.B. Garcia-Perez J.L. Moran J.V. The influence of LINE-1 and SINE retrotransposons on mammalian genomes.Microbiol. Spectr. 2015; 3 (MDNA3-0061-2014)Crossref Scopus (164) Google Scholar). ORF1 encodes a ∼40-kDa protein (ORF1p) with RNA-binding and nucleic acid chaperone activities (Hohjoh and Singer, 1996Hohjoh H. Singer M.F. Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA.EMBO J. 1996; 15: 630-639Crossref PubMed Scopus (285) Google Scholar, Khazina et al., 2011Khazina E. Truffault V. Büttner R. Schmidt S. Coles M. Weichenrieder O. Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition.Nat. Struct. Mol. Biol. 2011; 18: 1006-1014Crossref PubMed Scopus (101) Google Scholar, Martin and Bushman, 2001Martin S.L. Bushman F.D. Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon.Mol. Cell. Biol. 2001; 21: 467-475Crossref PubMed Scopus (285) Google Scholar). ORF2 encodes a ∼150-kDa protein (ORF2p) with endonuclease (EN) and reverse transcriptase (RT) activities (Doucet et al., 2010Doucet A.J. Hulme A.E. Sahinovic E. Kulpa D.A. Moldovan J.B. Kopera H.C. Athanikar J.N. Hasnaoui M. Bucheton A. Moran J.V. Gilbert N. Characterization of LINE-1 ribonucleoprotein particles.PLoS Genet. 2010; 6: e1001150Crossref PubMed Scopus (174) Google Scholar, Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, Mathias et al., 1991Mathias S.L. Scott A.F. Kazazian Jr., H.H. Boeke J.D. Gabriel A. Reverse transcriptase encoded by a human transposable element.Science. 1991; 254: 1808-1810Crossref PubMed Scopus (582) Google Scholar, Moran et al., 1996Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. High frequency retrotransposition in cultured mammalian cells.Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). L1 retrotransposition occurs by target-site primed reverse transcription (TPRT) (Cost et al., 2002Cost G.J. Feng Q. Jacquier A. Boeke J.D. Human L1 element target-primed reverse transcription in vitro.EMBO J. 2002; 21: 5899-5910Crossref PubMed Scopus (378) Google Scholar, Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, Luan et al., 1993Luan D.D. Korman M.H. Jakubczak J.L. Eickbush T.H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.Cell. 1993; 72: 595-605Abstract Full Text PDF PubMed Scopus (910) Google Scholar). The L1 5′ UTR sense-strand promoter (Athanikar et al., 2004Athanikar J.N. Badge R.M. Moran J.V. A YY1-binding site is required for accurate human LINE-1 transcription initiation.Nucleic Acids Res. 2004; 32: 3846-3855Crossref PubMed Scopus (130) Google Scholar, Swergold, 1990Swergold G.D. Identification, characterization, and cell specificity of a human LINE-1 promoter.Mol. Cell. Biol. 1990; 10: 6718-6729Crossref PubMed Scopus (344) Google Scholar) produces a full-length polyadenylated L1 transcript, which then is exported to the cytoplasm. ORF1p and ORF2p preferentially associate with their encoding transcript (Doucet et al., 2015Doucet A.J. Wilusz J.E. Miyoshi T. Liu Y. Moran J.V. A 3′ poly(A) tract is required for LINE-1 retrotransposition.Mol. Cell. 2015; 60: 728-741Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Esnault et al., 2000Esnault C. Maestre J. Heidmann T. Human LINE retrotransposons generate processed pseudogenes.Nat. Genet. 2000; 24: 363-367Crossref PubMed Scopus (639) Google Scholar, Wei et al., 2001Wei W. Gilbert N. Ooi S.L. Lawler J.F. Ostertag E.M. Kazazian H.H. Boeke J.D. Moran J.V. Human L1 retrotransposition: cis preference versus trans complementation.Mol. Cell. Biol. 2001; 21: 1429-1439Crossref PubMed Scopus (476) Google Scholar), leading to the formation of an L1 ribonucleoprotein particle (RNP) (Doucet et al., 2010Doucet A.J. Hulme A.E. Sahinovic E. Kulpa D.A. Moldovan J.B. Kopera H.C. Athanikar J.N. Hasnaoui M. Bucheton A. Moran J.V. Gilbert N. Characterization of LINE-1 ribonucleoprotein particles.PLoS Genet. 2010; 6: e1001150Crossref PubMed Scopus (174) Google Scholar, Hohjoh and Singer, 1996Hohjoh H. Singer M.F. Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA.EMBO J. 1996; 15: 630-639Crossref PubMed Scopus (285) Google Scholar, Kulpa and Moran, 2005Kulpa D.A. Moran J.V. Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition.Hum. Mol. Genet. 2005; 14: 3237-3248Crossref PubMed Scopus (135) Google Scholar, Martin, 1991Martin S.L. Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells.Mol. Cell. Biol. 1991; 11: 4804-4807Crossref PubMed Scopus (188) Google Scholar). Components of the L1 RNP gain nuclear access (Kubo et al., 2006Kubo S. Seleme M.C. Soifer H.S. Perez J.L. Moran J.V. Kazazian Jr., H.H. Kasahara N. L1 retrotransposition in nondividing and primary human somatic cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 8036-8041Crossref PubMed Scopus (142) Google Scholar), where L1 EN activity generates a single-strand endonucleolytic nick at a degenerate consensus sequence (5′-TTTTT/AA-3′ and variants of that sequence) in genomic DNA, generating a 3′-hydroxyl group (Cost and Boeke, 1998Cost G.J. Boeke J.D. Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure.Biochemistry. 1998; 37: 18081-18093Crossref PubMed Scopus (190) Google Scholar, Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, Flasch et al., 2019Flasch D.A. Macia Á. Sánchez L. Ljungman M. Heras S.R. García-Pérez J.L. Wilson T.E. Moran J.V. Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.Cell. 2019; 177: 837-851.e28Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, Sultana et al., 2019Sultana T. van Essen D. Siol O. Bailly-Bechet M. Philippe C. Zine El Aabidine A. Pioger L. Nigumann P. Saccani S. Andrau J.C. et al.The landscape of L1 retrotransposons in the human genome is shaped by pre-insertion sequence biases and post-insertion selection.Mol. Cell. 2019; 74: 555-570.e7Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Annealing between the thymidine residues liberated by L1 EN cleavage and L1 mRNA poly(A) tail provides a primer-template complex that the L1 RT uses to reverse transcribe (−) strand L1 cDNA from L1 RNA (Doucet et al., 2015Doucet A.J. Wilusz J.E. Miyoshi T. Liu Y. Moran J.V. A 3′ poly(A) tract is required for LINE-1 retrotransposition.Mol. Cell. 2015; 60: 728-741Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Kulpa and Moran, 2006Kulpa D.A. Moran J.V. Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles.Nat. Struct. Mol. Biol. 2006; 13: 655-660Crossref PubMed Scopus (209) Google Scholar, Monot et al., 2013Monot C. Kuciak M. Viollet S. Mir A.A. Gabus C. Darlix J.L. Cristofari G. The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.PLoS Genet. 2013; 9: e1003499Crossref PubMed Scopus (43) Google Scholar). Second-strand genomic DNA cleavage, (+) strand L1 cDNA synthesis, and L1 cDNA ligation to genomic DNA require elucidation but likely involve host-encoded proteins (Goodier, 2016Goodier J.L. Restricting retrotransposons: a review.Mob. DNA. 2016; 7: 16Crossref PubMed Scopus (240) Google Scholar, Pizarro and Cristofari, 2016Pizarro J.G. Cristofari G. Post-transcriptional control of LINE-1 retrotransposition by cellular host factors in somatic cells.Front. Cell Dev. Biol. 2016; 4: 14Crossref PubMed Scopus (53) Google Scholar). Cells have evolved mechanisms to restrict L1 retrotransposition (Levin and Moran, 2011Levin H.L. Moran J.V. Dynamic interactions between transposable elements and their hosts.Nat. Rev. Genet. 2011; 12: 615-627Crossref PubMed Scopus (392) Google Scholar, Pizarro and Cristofari, 2016Pizarro J.G. Cristofari G. Post-transcriptional control of LINE-1 retrotransposition by cellular host factors in somatic cells.Front. Cell Dev. Biol. 2016; 4: 14Crossref PubMed Scopus (53) Google Scholar). For example, L1 expression can be repressed by transcriptional and post-transcriptional mechanisms, and host factors involved in the innate immune response can destabilize L1 RNA, L1 cDNA, and/or L1-encoded proteins (Levin and Moran, 2011Levin H.L. Moran J.V. Dynamic interactions between transposable elements and their hosts.Nat. Rev. Genet. 2011; 12: 615-627Crossref PubMed Scopus (392) Google Scholar, Richardson et al., 2015Richardson S.R. Doucet A.J. Kopera H.C. Moldovan J.B. Garcia-Perez J.L. Moran J.V. The influence of LINE-1 and SINE retrotransposons on mammalian genomes.Microbiol. Spectr. 2015; 3 (MDNA3-0061-2014)Crossref Scopus (164) Google Scholar). Proteins involved in the DNA damage response and/or DNA repair also restrict L1 retrotransposition (Coufal et al., 2011Coufal N.G. Garcia-Perez J.L. Peng G.E. Marchetto M.C. Muotri A.R. Mu Y. Carson C.T. Macia A. Moran J.V. Gage F.H. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells.Proc. Natl. Acad. Sci. USA. 2011; 108: 20382-20387Crossref PubMed Scopus (176) Google Scholar, Gasior et al., 2008Gasior S.L. Roy-Engel A.M. Deininger P.L. ERCC1/XPF limits L1 retrotransposition.DNA Repair (Amst.). 2008; 7: 983-989Crossref PubMed Scopus (74) Google Scholar, Liu et al., 2018Liu N. Lee C.H. Swigut T. Grow E. Gu B. Bassik M.C. Wysocka J. Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators.Nature. 2018; 553: 228-232Crossref PubMed Scopus (141) Google Scholar, Servant et al., 2017Servant G. Streva V.A. Derbes R.S. Wijetunge M.I. Neeland M. White T.B. Belancio V.P. Roy-Engel A.M. Deininger P.L. The nucleotide excision repair pathway limits L1 retrotransposition.Genetics. 2017; 205: 139-153Crossref PubMed Scopus (24) Google Scholar). Proteins involved in DNA replication and/or DNA repair can facilitate L1 retrotransposition, but their mechanisms of action require elucidation (Benitez-Guijarro et al., 2018Benitez-Guijarro M. Lopez-Ruiz C. Tarnauskaitė Ž. Murina O. Mian Mohammad M. Williams T.C. Fluteau A. Sanchez L. Vilar-Astasio R. Garcia-Canadas M. et al.RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition.EMBO J. 2018; 37: 37Crossref Scopus (41) Google Scholar, Flasch et al., 2019Flasch D.A. Macia Á. Sánchez L. Ljungman M. Heras S.R. García-Pérez J.L. Wilson T.E. Moran J.V. Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.Cell. 2019; 177: 837-851.e28Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, Mita et al., 2018Mita P. Wudzinska A. Sun X. Andrade J. Nayak S. Kahler D.J. Badri S. LaCava J. Ueberheide B. Yun C.Y. et al.LINE-1 protein localization and functional dynamics during the cell cycle.eLife. 2018; 7: e30058Crossref PubMed Scopus (69) Google Scholar, Sultana et al., 2019Sultana T. van Essen D. Siol O. Bailly-Bechet M. Philippe C. Zine El Aabidine A. Pioger L. Nigumann P. Saccani S. Andrau J.C. et al.The landscape of L1 retrotransposons in the human genome is shaped by pre-insertion sequence biases and post-insertion selection.Mol. Cell. 2019; 74: 555-570.e7Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Suzuki et al., 2009Suzuki J. Yamaguchi K. Kajikawa M. Ichiyanagi K. Adachi N. Koyama H. Takeda S. Okada N. Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.PLoS Genet. 2009; 5: e1000461Crossref PubMed Scopus (96) Google Scholar, Taylor et al., 2013Taylor M.S. LaCava J. Mita P. Molloy K.R. Huang C.R. Li D. Adney E.M. Jiang H. Burns K.H. Chait B.T. et al.Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.Cell. 2013; 155: 1034-1048Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, Taylor et al., 2018Taylor M.S. Altukhov I. Molloy K.R. Mita P. Jiang H. Adney E.M. Wudzinska A. Badri S. Ischenko D. Eng G. et al.Dissection of affinity captured LINE-1 macromolecular complexes.eLife. 2018; 7: e30094Crossref PubMed Scopus (42) Google Scholar). Here, we conducted immunoprecipitation (IP)-coupled mass spectrometry analyses to identify L1 ORF2p interacting proteins. We demonstrate poly(ADP-ribose) (PAR) polymerase 2 (PARP2) is recruited to single-strand DNA (ssDNA) breaks generated by L1 EN cleavage. The subsequent enzymatic activation of PARP2 leads to the synthesis of PAR, which promotes the direct recruitment of the replication protein A (RPA) complex to sites of L1 integration. RPA can decrease the vulnerability of ssDNA intermediates generated during TPRT to APOBEC3A (A3A)-mediated cytidine deamination in vitro. However, A3A can associate with RPA, which may guide it to L1 integration sites. Thus, interactions among L1 ORF2p, activated PARP2, and RPA can facilitate retrotransposition, whereas the association of RPA with APOBEC3 proteins may, in principle, restrict L1 retrotransposition. We conducted IP-coupled mass spectrometry analyses to identify ORF2p interacting proteins. We constructed an L1 expression vector (pTMF3) that contains a T7 gene10 epitope tag and three tandem copies of a FLAG epitope tag at the C termini of ORF1p and ORF2p, respectively (Figure S1A). ORF2p-3FLAG co-precipitated with ORF1p-T7 (Figure S1B), and the epitope tags did not significantly affect retrotransposition efficiency (Figure S1C). Human ORF2p is expressed at lower levels than ORF1p (Doucet et al., 2010Doucet A.J. Hulme A.E. Sahinovic E. Kulpa D.A. Moldovan J.B. Kopera H.C. Athanikar J.N. Hasnaoui M. Bucheton A. Moran J.V. Gilbert N. Characterization of LINE-1 ribonucleoprotein particles.PLoS Genet. 2010; 6: e1001150Crossref PubMed Scopus (174) Google Scholar, McMillan and Singer, 1993McMillan J.P. Singer M.F. Translation of the human LINE-1 element, L1Hs.Proc. Natl. Acad. Sci. USA. 1993; 90: 11533-11537Crossref PubMed Scopus (66) Google Scholar, Taylor et al., 2013Taylor M.S. LaCava J. Mita P. Molloy K.R. Huang C.R. Li D. Adney E.M. Jiang H. Burns K.H. Chait B.T. et al.Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.Cell. 2013; 155: 1034-1048Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Thus, we generated an ORF2p-3FLAG monocistronic expression vector (pTMO2F3) to increase the production of ORF2p in HEK293T cells (Figure 1A). Expression of pTMO2F3 led to an increase in γ-H2AX levels, a well-known marker for double-stranded DNA (dsDNA) breaks (Hustedt and Durocher, 2016Hustedt N. Durocher D. The control of DNA repair by the cell cycle.Nat. Cell Biol. 2016; 19: 1-9Crossref PubMed Scopus (379) Google Scholar), when compared to a negative control lacking both L1 EN and L1 RT activities (Figure S1D). HEK293T cells were next transfected with pTMO2F3, an anti-FLAG antibody was used to purify the ORF2p-3FLAG complex, and associated host proteins were identified using mass spectrometry (Figure 1B). We identified ORF2p-3FLAG associated proteins that were absent (Table S1A) or significantly reduced (Table S1B) in controls. Some proteins were known to associate with L1 RNPs (e.g., PABPC1, PCNA, HSPA8, UPF1, and MOV10) (Dai et al., 2012Dai L. Taylor M.S. O’Donnell K.A. Boeke J.D. Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation.Mol. Cell. Biol. 2012; 32: 4323-4336Crossref PubMed Google Scholar, Goodier et al., 2013Goodier J.L. Cheung L.E. Kazazian Jr., H.H. Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition.Nucleic Acids Res. 2013; 41: 7401-7419Crossref PubMed Scopus (110) Google Scholar, Taylor et al., 2013Taylor M.S. LaCava J. Mita P. Molloy K.R. Huang C.R. Li D. Adney E.M. Jiang H. Burns K.H. Chait B.T. et al.Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.Cell. 2013; 155: 1034-1048Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar); others are involved in RNA metabolism and nuclear processes (Tables S1A and S1B). We conducted IP followed by western blot to validate a subset of the ORF2p-3FLAG interacting proteins (Figure 1C). ORF2p-3FLAG co-precipitated with PARP1, PARP2, KU80, H2B, SPT16, XPC, PCNA, RPA, HMCES, HUWE1, and SSBP1. Most of these proteins also associated at reduced levels with ORF2p-3FLAG expressed from a full-length RC-L1 (Figure 1C) (Doucet et al., 2010Doucet A.J. Hulme A.E. Sahinovic E. Kulpa D.A. Moldovan J.B. Kopera H.C. Athanikar J.N. Hasnaoui M. Bucheton A. Moran J.V. Gilbert N. Characterization of LINE-1 ribonucleoprotein particles.PLoS Genet. 2010; 6: e1001150Crossref PubMed Scopus (174) Google Scholar). ORF1p-1FLAG did not or only exhibited subtle interactions with the ORF2p-3FLAG-associated nuclear proteins (Figure 1C), suggesting that ORF1p is more abundant in cytoplasmic versus nuclear L1 RNPs or might form unstable complexes with ORF2p-associated nuclear proteins. Thus, in addition to the previously reported associations with PARP1, PCNA, RPA, and HMCES (Mita et al., 2018Mita P. Wudzinska A. Sun X. Andrade J. Nayak S. Kahler D.J. Badri S. LaCava J. Ueberheide B. Yun C.Y. et al.LINE-1 protein localization and functional dynamics during the cell cycle.eLife. 2018; 7: e30058Crossref PubMed Scopus (69) Google Scholar, Taylor et al., 2013Taylor M.S. LaCava J. Mita P. Molloy K.R. Huang C.R. Li D. Adney E.M. Jiang H. Burns K.H. Chait B.T. et al.Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.Cell. 2013; 155: 1034-1048Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, Taylor et al., 2018Taylor M.S. Altukhov I. Molloy K.R. Mita P. Jiang H. Adney E.M. Wudzinska A. Badri S. Ischenko D. Eng G. et al.Dissection of affinity captured LINE-1 macromolecular complexes.eLife. 2018; 7: e30094Crossref PubMed Scopus (42) Google Scholar), ORF2p interacts with at least seven other nuclear proteins. L1 EN activity is required for initiating TPRT (Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, Moran et al., 1996Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. High frequency retrotransposition in cultured mammalian cells.Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar). Because ssDNA breaks are sensed and repaired by two PAR polymerase family members (PARP1 and PARP2) (Gibson and Kraus, 2012Gibson B.A. Kraus W.L. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs.Nat. Rev. Mol. Cell Biol. 2012; 13: 411-424Crossref PubMed Scopus (855) Google Scholar), we used established assays to test whether a PARP inhibitor (PARPi), olaparib, affects L1 retrotransposition (Figure 2A; STAR Methods) (Gibson and Kraus, 2012Gibson B.A. Kraus W.L. New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs.Nat. Rev. Mol. Cell Biol. 2012; 13: 411-424Crossref PubMed Scopus (855) Google Scholar, Moran et al., 1996Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. High frequency retrotransposition in cultured mammalian cells.Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar, Ostertag et al., 2000Ostertag E.M. Prak E.T. DeBerardinis R.J. Moran J.V. Kazazian Jr., H.H. Determination of L1 retrotransposition kinetics in cultured cells.Nucleic Acids Res. 2000; 28: 1418-1423Crossref PubMed Scopus (222) Google Scholar, Wei et al., 2001Wei W. Gilbert N. Ooi S.L. Lawler J.F. Ostertag E.M. Kazazian H.H. Boeke J.D. Moran J.V. Human L1 retrotransposition: cis preference versus trans complementation.Mol. Cell. Biol. 2001; 21: 1429-1439Crossref PubMed Scopus (476) Google Scholar). L1 retrotransposition efficiency decreased significantly upon increasing the PARPi concentration (Figures 2B and S2A–S2C), and controls, in which HeLa-JVM cells were transfected with a plasmid expressing a blasticidin-resistant gene (pcDNA6), indicated that the reduction in retrotransposition efficiency was not due to PARPi toxicity (Figure 2B). We next examined whether altering PARP1 and/or PARP2 expression affects L1 retrotransposition (Figure S2D). Ectopic overexpression of PARP1 and/or PARP2 did not affect L1 retrotransposition. By comparison, short hairpin RNA (shRNA)-mediated knockdown of PARP1 or PARP2 in HEK293T cells led to a ∼50% reduction in L1 retrotransposition (Figures 2C and 2D). Mouse homozygous knockouts of either Parp1 or Parp2 are viable, whereas double knockouts lead to embryonic lethality, suggesting Parp1 and Parp2 may be functionally redundant (Ménissier de Murcia et al., 2003Ménissier de Murcia J. Ricoul M. Tartier L. Niedergang C. Huber A. Dantzer F. Schreiber V. Amé J.C. Dierich A. LeMeur M. et al.Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse.EMBO J. 2003; 22: 2255-2263Crossref PubMed Scopus (492) Google Scholar). Knockdown of both PARP1 and PARP2 in HEK293T cells led to an ∼80% reduction in L1 retrotransposition (Figures 2D and S2E), but treating these cells with PARPi did not further reduce retrotransposition (Figure S2F). Controls revealed that the reduction in L1 retrotransposition is not due to alterations in steady-state ORF2p levels or HEK293T survival rates (Figures S2G and S2H). L1 retrotransposition can occur by an alternative EN-independent pathway in cells that are p53-defective and contain mutations in genes critical for non-homologous end-joining (NHEJ) DNA repair, suggesting that L1 can exploit endogenous DNA lesions to initiate retrotransposition (Coufal et al., 2011Coufal N.G. Garcia-Perez J.L. Peng G.E. Marchetto M.C. Muotri A.R. Mu Y. Carson C.T. Macia A. Moran J.V. Gage F.H. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells.Proc. Natl. Acad. Sci. USA. 2011; 108: 20382-20387Crossref PubMed Scopus (176) Google Scholar, Kopera et al., 2011Kopera H.C. Moldovan J.B. Morrish T.A. Garcia-Perez J.L. Moran J.V. Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.Proc. Natl. Acad. Sci. USA. 2011; 108: 20345-20350Crossref PubMed Scopus (47) Google Scholar, Morrish et al., 2007Morrish T.A. Garcia-Perez J.L. Stamato T.D. Taccioli G.E. Sekiguchi J. Moran J.V. Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres.Nature. 2007; 446: 208-212Crossref PubMed Scopus (134) Google Scholar, Morrish et al., 2002Morrish T.A. Gilbert N. Myers J.S. Vincent B.J. Stamato T.D. Taccioli G.E. Batzer M.A. Moran J.V. DNA repair mediated by endonuclease-independent LINE-1 retrotransposition.Nat. Genet. 2002; 31: 159-165Crossref PubMed Scopus (347) Google Scholar). Thus, we transfected a NHEJ-defective Chinese hamster ovary (CHO) cell line (Li et al., 1995Li Z. Otevrel T. Gao Y. Cheng H.L. Seed B. Stamato T.D. Taccioli G.E. Alt F.W. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination.Cell. 1995; 83: 1079-1089Abstract Full Text PDF PubMed Scopus (398) Google Scholar) with a wild-type (WT) or EN-deficient L1 expression construct and observed efficient retrotransposition in the absence of the PARPi; however, increasing the PARPi concentration only led to a decrease in WT L1 retrotransposition (Figure 2E). Thus, PARP activity is important for canonical TPRT, but not EN-independent retrotransposition. PARP1 and PARP2 have different DNA-binding domains and are independently recruited to DNA breaks (Mortusewicz et al., 2007Mortusewicz O. Amé J.C. Schreiber V. Leonhardt H. Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells.Nucleic Acids Res. 2007; 35: 7665-7675Crossref PubMed Scopus (234) Google Scholar). Thus, we examined whether L1-induced ssDNA breaks trigger the recruitment of PARP1 and/or PARP2 to the ORF2p-3FLAG complex. We prepared WT, EN-deficient, RT-deficient, EN- and RT-deficient, or cysteine-rich (C)-domain-deficient ORF2p-3FLAG protein complexes (Figures 3A and 3B ). IP demonstrated that PARP1 and PABPC1 associated with the WT, EN-deficient, RT-deficient, and EN- and RT-deficient ORF2p-3FLAG proteins but only minimally associated with the C-domain-deficient protein (Figure 3B). In contrast, PARP2 only associated with WT and RT-deficient ORF2p (Figure 3B), indicating that L1 EN activity is critical for ORF2p-3FLAG/PARP2 association. SSBP1 and HMCES also exhibited a reduced ability to associate with the RT-deficient and EN- and RT-deficient ORF2p-3FLAG protein (Figure S3A), suggesting their association with ORF2p-3FLAG may be facilitated by ssDNAs generated during TPRT. We next tested whether L1 ORF2p-3FLAG directly interacts with PARP1 or PARP2 (Figure S3B). PARP1 interacted with an ORF2p-3FLAG derivative that lacks the EN domain; however, deleting the central regions of ORF2p (i.e., the Z or RT domains) led to reduced interactions; PARP2 did not associate with any of the ORF2p-3FLAG deletion derivatives (Figure S3B). PARP2 may associate with ORF2p after L1 EN nicks target site DNA. Thus, we treated the ORF2p-3FLAG IP complex with RNase or benzonase, a nuclease that can degrade RNA and DNA (Figure 3C). Benzonase treatment reduced the ORF2p-3FLAG/PARP2, but not the ORF2p-3FLAG/PARP1, interaction (Figure 3C). Controls revealed that RNase or benzonase treatment abolished the PABP" @default.
• W2970440112 created "2019-09-05" @default.
• W2970440112 creator A5016025349 @default.
• W2970440112 creator A5046950035 @default.
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• W2970440112 date "2019-09-01" @default.
• W2970440112 modified "2023-09-27" @default.
• W2970440112 title "Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition" @default.
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• W2970440112 doi "https://doi.org/10.1016/j.molcel.2019.07.018" @default.
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