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• W2443774396 abstract "•RECQL4 promotes 5′ end resection at DSBs•RECQL4 recruitment to DSBs depends on MRE11•RECQL4 promotes recruitment of CtIP to DSBs•RECQL4 helicase activity is required for 5′ DNA end resection The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5′ end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4’s helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4’s unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR. The RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5′ end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4’s helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4’s unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR. DNA double-strand breaks (DSBs) are generated by exogenous stress, endogenous replication, and programmed recombination events. Improperly repaired DSBs can lead to genome instability, chromosomal rearrangements, and/or cell death (Symington, 2014Symington L.S. End resection at double-strand breaks: mechanism and regulation.Cold Spring Harb. Perspect. Biol. 2014; 6 (a016436)Crossref PubMed Scopus (177) Google Scholar). DSBs are usually repaired by one of two major pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) (Aparicio et al., 2014Aparicio T. Baer R. Gautier J. DNA double-strand break repair pathway choice and cancer.DNA Repair (Amst.). 2014; 19: 169-175Crossref PubMed Scopus (221) Google Scholar). HR-dependent DSBR is mostly error free, but it requires a sister or non-sister chromatid as template and is only active during the S and G2 phases of the cell cycle. In contrast, NHEJ-dependent DSBR is error prone, DNA template-independent, and active during all phases of the cell cycle. HR-dependent DSBR is initiated by 5′ end resection of the DSBs, which generates 3′ protruding single-strand DNA (ssDNA) tails (Chen et al., 2013Chen H. Lisby M. Symington L.S. RPA coordinates DNA end resection and prevents formation of DNA hairpins.Mol. Cell. 2013; 50: 589-600Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Zhu et al., 2008Zhu Z. Chung W.H. Shim E.Y. Lee S.E. Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.Cell. 2008; 134: 981-994Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). RPA coats the ssDNA, and then RAD51 replaces RPA to promote strand invasion. This is followed by repair synthesis, dissolution, and resolution of Holliday junctions and ligation of the ends (Prakash et al., 2015Prakash R. Zhang Y. Feng W. Jasin M. Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins.Cold Spring Harb. Perspect. Biol. 2015; 7: a016600Crossref Scopus (479) Google Scholar). It is generally considered that DNA end resection occurs in two steps (Cejka et al., 2010Cejka P. Cannavo E. Polaczek P. Masuda-Sasa T. Pokharel S. Campbell J.L. Kowalczykowski S.C. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.Nature. 2010; 467: 112-116Crossref PubMed Scopus (344) Google Scholar, Gravel et al., 2008Gravel S. Chapman J.R. Magill C. Jackson S.P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.Genes Dev. 2008; 22: 2767-2772Crossref PubMed Scopus (453) Google Scholar, Mimitou and Symington, 2008Mimitou E.P. Symington L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.Nature. 2008; 455: 770-774Crossref PubMed Scopus (767) Google Scholar, Nimonkar et al., 2011Nimonkar A.V. Genschel J. Kinoshita E. Polaczek P. Campbell J.L. Wyman C. Modrich P. Kowalczykowski S.C. BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.Genes Dev. 2011; 25: 350-362Crossref PubMed Scopus (498) Google Scholar, Niu et al., 2010Niu H. Chung W.H. Zhu Z. Kwon Y. Zhao W. Chi P. Prakash R. Seong C. Liu D. Lu L. et al.Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.Nature. 2010; 467: 108-111Crossref PubMed Scopus (289) Google Scholar, Zhu et al., 2008Zhu Z. Chung W.H. Shim E.Y. Lee S.E. Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.Cell. 2008; 134: 981-994Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). The first step is the initial resection by Mre11-Rad50-Xrs2 (MRX) and Sae2 at the DSB in yeast (Cannavo and Cejka, 2014Cannavo E. Cejka P. Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks.Nature. 2014; 514: 122-125Crossref PubMed Scopus (278) Google Scholar, Mimitou and Symington, 2008Mimitou E.P. Symington L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.Nature. 2008; 455: 770-774Crossref PubMed Scopus (767) Google Scholar) or by MRE11-RAD50-NBS1 (MRN) and CtIP (CtBP-interacting protein) in human cells (Sartori et al., 2007Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (991) Google Scholar, You et al., 2009You Z. Shi L.Z. Zhu Q. Wu P. Zhang Y.W. Basilio A. Tonnu N. Verma I.M. Berns M.W. Hunter T. CtIP links DNA double-strand break sensing to resection.Mol. Cell. 2009; 36: 954-969Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This is followed by extensive resection by either exonuclease1 (EXO1) or DNA2/BLM/TOP3/RMI1/2 (Dna2/Sgs1/Top3/Rmi1 in yeast) (Cejka et al., 2010Cejka P. Cannavo E. Polaczek P. Masuda-Sasa T. Pokharel S. Campbell J.L. Kowalczykowski S.C. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.Nature. 2010; 467: 112-116Crossref PubMed Scopus (344) Google Scholar, Gravel et al., 2008Gravel S. Chapman J.R. Magill C. Jackson S.P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.Genes Dev. 2008; 22: 2767-2772Crossref PubMed Scopus (453) Google Scholar, Mimitou and Symington, 2008Mimitou E.P. Symington L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.Nature. 2008; 455: 770-774Crossref PubMed Scopus (767) Google Scholar, Nimonkar et al., 2008Nimonkar A.V. Ozsoy A.Z. Genschel J. Modrich P. Kowalczykowski S.C. Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair.Proc. Natl. Acad. Sci. USA. 2008; 105: 16906-16911Crossref PubMed Scopus (243) Google Scholar, Nimonkar et al., 2011Nimonkar A.V. Genschel J. Kinoshita E. Polaczek P. Campbell J.L. Wyman C. Modrich P. Kowalczykowski S.C. BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.Genes Dev. 2011; 25: 350-362Crossref PubMed Scopus (498) Google Scholar, Niu et al., 2010Niu H. Chung W.H. Zhu Z. Kwon Y. Zhao W. Chi P. Prakash R. Seong C. Liu D. Lu L. et al.Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.Nature. 2010; 467: 108-111Crossref PubMed Scopus (289) Google Scholar, Zhu et al., 2008Zhu Z. Chung W.H. Shim E.Y. Lee S.E. Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.Cell. 2008; 134: 981-994Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). RECQL4 is one of five RecQ helicase proteins in mammalian cells. Defects in human RECQL4 are associated with three genetic diseases: Rothmund-Thomson syndrome (RTS), RAPADILINO, and Baller-Gerold syndrome (Siitonen et al., 2009Siitonen H.A. Sotkasiira J. Biervliet M. Benmansour A. Capri Y. Cormier-Daire V. Crandall B. Hannula-Jouppi K. Hennekam R. Herzog D. et al.The mutation spectrum in RECQL4 diseases.Eur. J. Hum. Genet. 2009; 17: 151-158Crossref PubMed Scopus (153) Google Scholar) as well as several cancers (Fang et al., 2013Fang H. Nie L. Chi Z. Liu J. Guo D. Lu X. Hei T.K. Balajee A.S. Zhao Y. RecQL4 helicase amplification is involved in human breast tumorigenesis.PLoS ONE. 2013; 8: e69600Crossref PubMed Scopus (35) Google Scholar, Lu et al., 2014bLu L. Jin W. Liu H. Wang L.L. RECQ DNA helicases and osteosarcoma.Adv. Exp. Med. Biol. 2014; 804: 129-145Crossref PubMed Scopus (28) Google Scholar, Su et al., 2010Su Y. Meador J.A. Calaf G.M. Proietti De-Santis L. Zhao Y. Bohr V.A. Balajee A.S. Human RecQL4 helicase plays critical roles in prostate carcinogenesis.Cancer Res. 2010; 70: 9207-9217Crossref PubMed Scopus (51) Google Scholar). It is well established that RECQL4 is required for the assembly of the DNA replication initiation machinery (Im et al., 2009Im J.S. Ki S.H. Farina A. Jung D.S. Hurwitz J. Lee J.K. Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins.Proc. Natl. Acad. Sci. USA. 2009; 106: 15628-15632Crossref PubMed Scopus (141) Google Scholar, Sangrithi et al., 2005Sangrithi M.N. Bernal J.A. Madine M. Philpott A. Lee J. Dunphy W.G. Venkitaraman A.R. Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome.Cell. 2005; 121: 887-898Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Xu et al., 2009Xu X. Rochette P.J. Feyissa E.A. Su T.V. Liu Y. MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication.EMBO J. 2009; 28: 3005-3014Crossref PubMed Scopus (106) Google Scholar). However, the role of RECQL4 in DNA repair is less clear (Croteau et al., 2014Croteau D.L. Popuri V. Opresko P.L. Bohr V.A. Human RecQ helicases in DNA repair, recombination, and replication.Annu. Rev. Biochem. 2014; 83: 519-552Crossref PubMed Scopus (377) Google Scholar). Lack of RECQL4 increases persistent DNA damage and triggers cellular senescence in human and mouse primary fibroblasts (Lu et al., 2014aLu H. Fang E.F. Sykora P. Kulikowicz T. Zhang Y. Becker K.G. Croteau D.L. Bohr V.A. Senescence induced by RECQL4 dysfunction contributes to Rothmund-Thomson syndrome features in mice.Cell Death Dis. 2014; 5: e1226Crossref PubMed Scopus (39) Google Scholar). RECQL4 is recruited to laser-induced DSBs and RTS fibroblasts are sensitive to ionizing radiation (IR), suggesting that RECQL4 plays a role in DSBR (Singh et al., 2010Singh D.K. Karmakar P. Aamann M. Schurman S.H. May A. Croteau D.L. Burks L. Plon S.E. Bohr V.A. The involvement of human RECQL4 in DNA double-strand break repair.Aging Cell. 2010; 9: 358-371Crossref PubMed Scopus (72) Google Scholar). Recently, we showed that depletion of RECQL4 inhibits NHEJ in U2OS cells (Shamanna et al., 2014Shamanna R.A. Singh D.K. Lu H. Mirey G. Keijzers G. Salles B. Croteau D.L. Bohr V.A. RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex.Carcinogenesis. 2014; 35: 2415-2424Crossref PubMed Scopus (42) Google Scholar). Nevertheless, RECQL4 is highly expressed during S phase (Singh et al., 2012Singh D.K. Popuri V. Kulikowicz T. Shevelev I. Ghosh A.K. Ramamoorthy M. Rossi M.L. Janscak P. Croteau D.L. Bohr V.A. The human RecQ helicases BLM and RECQL4 cooperate to preserve genome stability.Nucleic Acids Res. 2012; 40: 6632-6648Crossref PubMed Scopus (38) Google Scholar, Xu et al., 2009Xu X. Rochette P.J. Feyissa E.A. Su T.V. Liu Y. MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication.EMBO J. 2009; 28: 3005-3014Crossref PubMed Scopus (106) Google Scholar), when HR-dependent DSBR dominates. Thus, we explore the possibility that RECQL4 also plays a role in HR-dependent DSBR. We find that RECQL4 promotes DNA end resection and HR-dependent DSBR by stimulating the association of CtIP with MRN at DSBs and that the helicase activity of RECQL4 is necessary for DNA end resection. Together, these findings suggest that RECQL4 plays an important role in the DNA end resection step of HR-mediated DSBR in human cells. Endogenous RECQL4 co-localized with γH2AX at laser-induced DSBs in U2OS cells (Figure 1A) and depletion of RECQL4 caused U2OS and HeLa cells to be significantly more sensitive to IR (Figures 1B and S1A). Since DSB repair pathway choice is cell-cycle regulated (Aparicio et al., 2014Aparicio T. Baer R. Gautier J. DNA double-strand break repair pathway choice and cancer.DNA Repair (Amst.). 2014; 19: 169-175Crossref PubMed Scopus (221) Google Scholar), we first examined the effect of RECQL4 depletion on cell-cycle progression. Knockdown of RECQL4 did not perturb cell-cycle progression significantly in U2OS or HEK293T cells and did not alter expression of cell-cycle marker proteins Cyclin A and Cyclin D1 (Figure S2), which is consistent with a previous finding in HEK293 cells (Park et al., 2006Park S.J. Lee Y.J. Beck B.D. Lee S.H. A positive involvement of RecQL4 in UV-induced S-phase arrest.DNA Cell Biol. 2006; 25: 696-703Crossref PubMed Scopus (26) Google Scholar). The role of RECQL4 in HR-dependent DSBR was then investigated in DR-GFP U2OS cells, which can be scored for efficiency of HR-mediated repair of an I-SceI endonuclease-induced DSB by measuring the fraction of GFP-positive cells (Pierce et al., 1999Pierce 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, Wang et al., 2014Wang Q. Goldstein M. Alexander P. Wakeman T.P. Sun T. Feng J. Lou Z. Kastan M.B. Wang X.F. Rad17 recruits the MRE11-RAD50-NBS1 complex to regulate the cellular response to DNA double-strand breaks.EMBO J. 2014; 33: 862-877Crossref PubMed Scopus (60) Google Scholar). DR-GFP U2OS cells were transfected with one of three RECQL4-targeted small interfering RNAs (siRNAs). The efficiency of RECQL4 knockdown was about 90%, 60%, and 50% for siRQ4, siRQ4-2, and siRQ4-3, respectively (Figure 1D). Given that siRQ4 produced the greatest knockdown it was used in all subsequent experiments. Depletion of RECQL4 by siRQ4 significantly reduced the proportion of GFP-positive cells by 73%, from 5.5% in control cells to 1.47% in knockdown cells (Figures 1C and 1D), suggesting that RECQL4 plays a crucial role in HR-dependent DSBR. The other two siRNAs, siRQ4-2 and siRQ4-3, also significantly reduced the proportions of GFP-positive cells to 3.71% and 4.0%, respectively (Figures 1C and 1D). These data show that knockdown efficiency of RECQL4 correlates with a decrease of HR-mediated DSBR. RECQL4 is rapidly recruited to laser-induced DSBs where it is retained for a short time (Singh et al., 2010Singh D.K. Karmakar P. Aamann M. Schurman S.H. May A. Croteau D.L. Burks L. Plon S.E. Bohr V.A. The involvement of human RECQL4 in DNA double-strand break repair.Aging Cell. 2010; 9: 358-371Crossref PubMed Scopus (72) Google Scholar). Thus, we speculated that it plays a role in the early stages of HR repair. As mentioned above, the first step of HR-mediated DSBR is 5′-3′ end resection of the DSBs to generate 3′ protruding ssDNA tails, which are rapidly coated by RPA to form a nuclease-resistant protective protein-DNA filament (Chen et al., 2013Chen H. Lisby M. Symington L.S. RPA coordinates DNA end resection and prevents formation of DNA hairpins.Mol. Cell. 2013; 50: 589-600Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). We then examined RPA foci formation in RECQL4-depleted cells. After exposure to 10 Gy of IR, the fraction of cells with >20 RPA foci was ∼33.6% in the siRQ4-treated U2OS cells, significantly less than 75.8% in the control cells (Figure 1E). Depletion of RECQL4 also repressed RPA foci formation after IR in HeLa cells (Figure S1B). Consistent with these results, the abundance of phosphorylated RPA32 on serine 4 and serine 8, a maker of ssDNA-bound RPA (Shao et al., 1999Shao R.G. Cao C.X. Zhang H. Kohn K.W. Wold M.S. Pommier Y. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes.EMBO J. 1999; 18: 1397-1406Crossref PubMed Scopus (304) Google Scholar), was increased in IR-treated control U2OS and HEK293T cells, but not in RECQL4 knockdown cells (Figures 1F and S1C). AID-DIvA U2OS cells have been used to directly quantify ssDNA generated by 5′ end resection at two AsiSI-induced DSBs (Aymard et al., 2014Aymard F. Bugler B. Schmidt C.K. Guillou E. Caron P. Briois S. Iacovoni J.S. Daburon V. Miller K.M. Jackson S.P. Legube G. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.Nat. Struct. Mol. Biol. 2014; 21: 366-374Crossref PubMed Scopus (406) Google Scholar). With addition of 4-hydroxytamoxifen (4-OHT), the AsiSI endonuclease fused to an estrogen receptor ligand binding domain translocates from the cytoplasm to the nucleus to induce DSBs (Aymard et al., 2014Aymard F. Bugler B. Schmidt C.K. Guillou E. Caron P. Briois S. Iacovoni J.S. Daburon V. Miller K.M. Jackson S.P. Legube G. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.Nat. Struct. Mol. Biol. 2014; 21: 366-374Crossref PubMed Scopus (406) Google Scholar). Genomic DNA from these cells was prepared and analyzed for ssDNA at two DSBs by TaqMan qPCR, as previously described (Aymard et al., 2014Aymard F. Bugler B. Schmidt C.K. Guillou E. Caron P. Briois S. Iacovoni J.S. Daburon V. Miller K.M. Jackson S.P. Legube G. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.Nat. Struct. Mol. Biol. 2014; 21: 366-374Crossref PubMed Scopus (406) Google Scholar, Zhou et al., 2014Zhou Y. Caron P. Legube G. Paull T.T. Quantitation of DNA double-strand break resection intermediates in human cells.Nucleic Acids Res. 2014; 42: e19Crossref PubMed Scopus (151) Google Scholar). Consistent with a previous report (Zhou et al., 2014Zhou Y. Caron P. Legube G. Paull T.T. Quantitation of DNA double-strand break resection intermediates in human cells.Nucleic Acids Res. 2014; 42: e19Crossref PubMed Scopus (151) Google Scholar), we observed notably lower ssDNA in cells treated with CtIP siRNA because CtIP stimulates DNA end resection (Sartori et al., 2007Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (991) Google Scholar). On the contrary, depletion of 53BP1 increased the amount of ssDNA (Figure 1G), since 53BP1 inhibits resection (Bunting et al., 2010Bunting S.F. Callén E. Wong N. Chen H.T. Polato F. Gunn A. Bothmer A. Feldhahn N. Fernandez-Capetillo O. Cao L. et al.53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.Cell. 2010; 141: 243-254Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar). To investigate the role of RECQL4 in 5′ end resection, ssDNA content was measured in siRQ4-transfected AID-DIvA U2OS cells. Interestingly, the amount of ssDNA generated at position 335 nt from DSB1 was 40.2% lower than that in siCtrl-treated cells, and similar to the reduction caused by CtIP depletion. At DSB2, depletion of RECQL4 reduced ssDNA content by 57.2%, 70.1%, and 75.2% at positions 364, 1,754, and 3,574 nt, respectively. Together, these results demonstrate that RECQL4 is important for HR by promoting 5′ end resection of DSBs. To explore the function of RECQL4 in 5′ end resection, we used mass spectroscopy to analyze proteins captured by co-immunoprecipitation (IP) with RECQL4-3xFLAG from irradiated HEK293T cells in the presence of benzonase. MRN components MRE11 and RAD50 and other DNA resection proteins BLM, EXO1, and DNA2 were identified (Figure S3A; Table S1), and their interactions with RECQL4 were independently confirmed by IP with GFP-RECQL4 in the presence of benzonase (Figure S3B) or ethidium bromide (Figure S3C). We found that RECQL4 co-localized with MRE11 at DSBs and that the RECQL4-MRN interaction is stimulated by IR (Figures 2A and 2B ). Purified recombinant RECQL4 also immunoprecipitated recombinant MRE11, RAD50, and NBS1 (Figure 2C), indicating complex formation between RECQL4 and MRN. To map the interaction region of RECQL4 with MRE11, purified RECQL4-3xFLAG and truncation fragments were incubated with purified YFP-MRE11 bound to GFP agarose beads. YFP-MRE11 pulled down full-length and the N-terminal domain of RECQL4 (Figure 2D), indicating that the N-terminal fragment of RECQL4 is responsible for the interaction with MRE11. To determine whether the interaction between RECQL4 and MRN is functional, we measured the nuclease activity of MRN on closed-circular single-strand PhiX174 DNA in the presence of RECQL4 in vitro as previously reported (Sartori et al., 2007Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (991) Google Scholar). Wild-type MRN and nuclease-dead MRN-ND (H129L/D130V) (Stracker et al., 2002Stracker T.H. Carson C.T. Weitzman M.D. Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex.Nature. 2002; 418: 348-352Crossref PubMed Scopus (417) Google Scholar) as well as RECQL4 and its helicase-dead mutant RQ4KM were used (Figure S4A). We found that RECQL4 slightly stimulated the nuclease activity of MRN on closed-circular single-strand PhiX174 DNA (Figure 2E). Both RECQL4 and MRE11 are rapidly recruited to DSB (Haince et al., 2008Haince J.F. McDonald D. Rodrigue A. Déry U. Masson J.Y. Hendzel M.J. Poirier G.G. PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites.J. Biol. Chem. 2008; 283: 1197-1208Crossref PubMed Scopus (405) Google Scholar, Singh et al., 2010Singh D.K. Karmakar P. Aamann M. Schurman S.H. May A. Croteau D.L. Burks L. Plon S.E. Bohr V.A. The involvement of human RECQL4 in DNA double-strand break repair.Aging Cell. 2010; 9: 358-371Crossref PubMed Scopus (72) Google Scholar), and thus we evaluated whether RECQL4 and MRE11 affected each other’s recruitment to DNA damage. GFP-RECQL4 was recruited significantly less to laser-induced DSBs in siMRE11-treated U2OS cells than in control cells (Figure 2F), and there is less chromatin-bound RECQL4 in siMRE11-treated U2OS cells than in control cells after IR (Figure S4B). However, depletion of RECQL4 did not affect the recruitment of YFP-MRE11 to laser-induced DSB (Figure S4C), suggesting that recruitment of RECQL4 to DSBs requires MRE11, but not vice versa. Given that MRE11 nuclease regulates the pathway choice between NHEJ and HR (Shibata et al., 2014Shibata A. Moiani D. Arvai A.S. Perry J. Harding S.M. Genois M.M. Maity R. van Rossum-Fikkert S. Kertokalio A. Romoli F. et al.DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities.Mol. Cell. 2014; 53: 7-18Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), the dynamics of GFP-RECQL4 recruitment was also evaluated in cells exposed to mirin, which specifically inhibits the MRE11 exonuclease but does not inhibit MRN complex formation (Dupré et al., 2008Dupré A. Boyer-Chatenet L. Sattler R.M. Modi A.P. Lee J.H. Nicolette M.L. Kopelovich L. Jasin M. Baer R. Paull T.T. Gautier J. A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex.Nat. Chem. Biol. 2008; 4: 119-125Crossref PubMed Scopus (291) Google Scholar). RECQL4 was still rapidly recruited to laser-induced DSBs in mirin-treated cells (Figure 2G), indicating that the recruitment of RECQL4 does not depend on the exonuclease activity of MRE11. However, GFP-RECQL4 was retained at DSBs for a significantly shorter time after mirin treatment (Figure 2G). This suggests that retention of RECQL4 at DSBs is regulated by MRE11 nuclease activity. When ssDNA was measured at AsiSI-induced DSB in AID-DIvA U2OS cells pre-treated with mirin, siRQ4 or siMRE11, a lower amount of ssDNA was detected (Figure 2H). However, the effect was not additive (Figure 2H). Using the DR-GFP reporter system, it was observed that pre-treatment with siRQ4 or siMRE11 significantly reduced HR-mediated DSBR, but the effect was also not additive (Figure 2I). These results suggest that RECQL4 functions downstream of MRN to promote DNA 5′ end resection and HR-dependent DSBR. CtIP is required for initiation of MRN-catalyzed 5′ end resection at DSBs (Chen et al., 2008Chen L. Nievera C.J. Lee A.Y. Wu X. Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair.J. Biol. Chem. 2008; 283: 7713-7720Crossref PubMed Scopus (321) Google Scholar, Sartori et al., 2007Sartori A.A. Lukas C. Coates J. Mistrik M. Fu S. Bartek J. Baer R. Lukas J. Jackson S.P. Human CtIP promotes DNA end resection.Nature. 2007; 450: 509-514Crossref PubMed Scopus (991) Google Scholar, Yuan and Chen, 2009Yuan J. Chen J. N terminus of CtIP is critical for homologous recombination-mediated double-strand break repair.J. Biol. Chem. 2009; 284: 31746-31752Crossref PubMed Scopus (57) Google Scholar). Here, we found that RECQL4 co-localized with CtIP at laser-induced DSBs (Figure 3A) and interacted with CtIP in irradiated HEK293T cells (Figures 3B, S3B, and S3C). The interaction between CtIP and RECQL4 appeared to be stronger in IR-treated cells (Figure 3B). CoIP of recombinant RECQL4 and CtIP suggests that RECQL4 interacts directly with CtIP (Figure 3C), and the N terminus of RECQL4 was mapped as the interacting region with CtIP (Figure 3D). Recruitment of RECQL4 reaches its peak about 1 min after laser damage (Figure 2G), while CtIP needs much longer (Wang et al., 2013Wang H. Shi L.Z. Wong C.C. Han X. Hwang P.Y. Truong L.N. Zhu Q. Shao Z. Chen D.J. Berns M.W. et al.The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.PLoS Genet. 2013; 9: e1003277Crossref PubMed Scopus (164) Google Scholar). Considering the direct interaction between RECQL4 and CtIP, it is possible that RECQL4 promotes CtIP recruitment to DSBs. To test this hypothesis, we first measured the abundance of chromatin-bound CtIP in control and RECQL4 knockdown U2OS cells after IR and found that IR increased chromatin-bound CtIP in the control cells but not in RECQL4-depleted cells (Figure 3E). Interestingly, more mobility shift of chromatin-bound CtIP was detected in control cells that in RECQL4-depleted cells after IR (Figure 3E), indicating that RECQL4 promotes IR-induced posttranslational modification of CtIP. Also, IR-treated control U2OS cells had an average of 22.6 GFP-CtIP foci per cell, significantly higher than that in siRQ4-treated cells (Figure 3F). Furthermore, in the RECQL4 knockdown U2OS cells, recruitment of GFP-CtIP was significantly slower and less efficient than that in control cells (Figure 3G). Together these data suggest that RECQL4 promotes stable CtIP recruitment to DSBs. Given that RECQL4 promotes recruitment of CtIP to DSBs, we asked whether RECQL4 is required for MRN-CtIP complex formation after IR. Pull-down assays were conducted in control and RECQL4 knockdown HEK293T cells expressing YFP-MRE11 or GFP-CtIP. Expression levels of MRE11, RAD50, NBS1, and CtIP proteins were similar in control and RECQL4 knockdown cells (input of Figure 3H). Cell-cycle status was not significantly different between RECQL4-depleted and control HEK293T cells (Figures S2D and S2E). IP of YFP-MRE11 efficiently pulled down similar amounts of RAD50 and NBS1 from control and RECQL4 knockdown cells. In contrast, the interaction between MRE11 and CtIP was inhibited by knockdown of RECQL4 (Figure 3H). In the reverse experiments with GFP-CtIP-expressing cells, GFP-CtIP efficiently co-immunoprecipitated MRE11, RAD50, and NBS1 from control cells but much less from RECQL4 knockdown cells (Figure 3H). These data are consistent with the idea that RECQL4 promotes the interaction between MRN and CtIP in human cells. Depletion of RECQL4 or CtIP significantly reduced ssDNA generation at DSB1 in AID-DIvA cells (Figure 3I). However, there were no differences among RECQL4 or CtIP-depleted cells and RECQL4/CtIP double-knockdown cells. A similar result was obtained from the experiments measuring the HR efficiency (Figure 3J). These results imply that RECQL4 and CtIP both play a role in HR-dependent DSBR and that RECQL4 promotes recruitment of CtIP to DSBs. 5′ resection, initiated by the MRN-CtIP complex, is extended by BLM/DNA2 and EXO1 via two alternative pathways (Cejka, 2015Cejka P. DNA end resection: nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 229" @default.
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• W2443774396 date "2016-06-01" @default.
• W2443774396 modified "2023-10-17" @default.
• W2443774396 title "RECQL4 Promotes DNA End Resection in Repair of DNA Double-Strand Breaks" @default.
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• W2443774396 doi "https://doi.org/10.1016/j.celrep.2016.05.079" @default.
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