Cell Cycle-dependent Complex Formation of BRCA1·CtIP·MRN Is Important for DNA Double-strand Break Repair
BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1·CtIP·MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G2 phases of the cell cycle. BRCA1 plays an important role in the homologous recombination (HR)-mediated DNA double-strand break (DSB) repair, but the mechanism is not clear. Here we describe that BRCA1 forms a complex with CtIP and MRN (Mre11/Rad50/Nbs1) in a cell cycle-dependent manner. Significantly, the complex formation, especially the ionizing radiation-enhanced association of BRCA1 with MRN, requires cyclin-dependent kinase activity. CtIP directly interacts with Nbs1. The in vivo association of BRCA1 with MRN is largely dependent on the association of CtIP with the BRCT domains at the C terminus of BRCA1, whereas the N terminus of BRCA1 also contributes to its association with MRN. CtIP, as well as the interaction of BRCA1 with CtIP and MRN, is critical for IR-induced single-stranded DNA formation and cellular resistance to radiation. Consistently, CtIP itself is required for efficient HR-mediated DSB repair, like BRCA1 and MRN. These studies suggest that the complex formation of BRCA1·CtIP·MRN is important for facilitating DSB resection to generate single-stranded DNA that is needed for HR-mediated DSB repair. Because cyclin-dependent kinase is important for establishing IR-enhanced interaction of MRN with BRCA1, we propose that the cell cycle-dependent complex formation of BRCA1, CtIP, and MRN contributes to the activation of HR-mediated DSB repair in the S and G2 phases of the cell cycle. Individuals carrying germ line mutations in BRCA1 have an extremely high risk of developing breast or ovarian cancer (1Nathanson K.L. Wooster R. Weber B.L. Nat. Med. 2001; 7: 552-556Crossref PubMed Scopus (372) Google Scholar). Compelling evidence suggests that BRCA1 plays important roles in the maintenance of genome stability (2Scully R. Livingston D.M. Nature. 2000; 408: 429-432Crossref PubMed Scopus (556) Google Scholar, 3Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar). Although BRCA1 possesses multiple cellular functions including checkpoint control, transcription regulation, ubiquitination, apoptosis, and DNA repair (3Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar, 4Starita L.M. Parvin J.D. Curr. Opin. 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Genes Dev. 2006; 20: 34-46Crossref PubMed Scopus (258) Google Scholar). the of the BRCA1·CtIP·MRN we the interaction of BRCA1 with MRN the cell cycle and DNA The interaction of BRCA1 and CtIP is cell cycle-dependent through the of the BRCT of BRCA1 with CtIP (26Yu X. Chen J. Mol. Cell. Biol. 2004; 24: 9478-9486Crossref PubMed Scopus (312) Google Scholar). by and found that MRN was with BRCA1 when the S and G2 phases of the cell cycle in a as the association of BRCA1 with CtIP and not These suggest that BRCA1, CtIP, and MRN a protein complex when the S and G2 phases of the cell cycle. The interaction of BRCA1 and is by when are in to the X. Rathbun G. Lane W.S. Weaver D.T. Livingston D.M. Cold Spring Harbor Symp. Quant. Biol. 2000; 65: 535-545Crossref PubMed Scopus (7) Google Scholar, R.A. Sobhian B. Pathania S. Cantor S.B. Nakatani Y. Livingston D.M. Genes Dev. 2006; 20: 34-46Crossref PubMed Scopus (258) Google Scholar). 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Chen P.L. Subramanian T. Chinnadurai G. Tomlinson G. Osborne C.K. Sharp Z.D. Lee W.H. J. Biol. Chem. 1999; 274: 11334-11338Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). as in and not the interaction of BRCA1 with MRN is in the BRCA1 cell line in its cell line with BRCA1 R. S. K. Chen J. M. Livingston D.M. Mol. Cell. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). a BRCA1 for one of the BRCT and suggest that the of BRCA1 is important for BRCA1 to with MRN. the BRCT domains are required for BRCA1 to with CtIP, the that the interaction of BRCA1 with CtIP is needed for efficient interaction of BRCA1 with MRN. CtIP directly with MRN, we with and Mre11 as well as that but not directly interacts with CtIP also the interaction of and with a of CtIP and and CtIP with can directly with CtIP in the of BRCA1 in and a that CtIP the interaction of BRCA1 with MRN. Although can directly with CtIP in the in vivo interaction of with CtIP is significantly when forms a complex with Mre11 and of and to the interaction of with not with and the with The association of the with CtIP is that of These suggest that the MRN complex is important for an efficient association of MRN with CtIP in can directly with Because BRCA1 is present in interaction of MRN with CtIP be to a complex formation of the N and C of BRCA1 for of BRCA1, CtIP, and the BRCT domains are required for BRCA1 to with CtIP and MRN in we the BRCA1 is to MRN. The was with cell not MRN cell interacts with CtIP These suggest that CtIP is important for the interaction of BRCA1 with MRN in is not to the interaction of MRN with the BRCA1 of BRCA1 in to the CtIP BRCT be required for BRCA1 to a complex with MRN. was that the of BRCA1 interacts with MRN in vivo X. Rathbun G. Lane W.S. Weaver D.T. Livingston D.M. Cold Spring Harbor Symp. Quant. Biol. 2000; 65: 535-545Crossref PubMed Scopus (7) Google Scholar). that the interacts with in These suggest that the N terminus of BRCA1 also directly with and interaction to complex formation of Consistently, a BRCA1 for the is significantly in its interaction with MRN studies suggest that BRCA1 interacts with MRN through both the N and C of BRCA1 and CtIP the C terminus interaction BRCA1 and and of BRCA1 are important for complex formation of BRCA1, CtIP, and MRN in of CtIP to a in DSB both BRCA1 and MRN in DSB repair, association of CtIP with BRCA1 and MRN to CtIP is also important for DSB repair. the of CtIP in by of in CtIP K. S. S. S. R.A. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of CtIP to phosphorylation as X. Chen J. Mol. Cell. Biol. 2004; 24: 9478-9486Crossref PubMed Scopus (312) Google and not a in cell when the of CtIP was by the we used not However, of CtIP to a of These suggest that CtIP an important role in DNA DSB repair. directly the role of CtIP in DSB repair, we the well DSB repair to DSBs in Jasin M. 2001; PubMed Google Scholar). homologous recombination of and in a was A.J. Jasin M. Genes Dev. 1999; PubMed Scopus Google and In of a DSB and when a DSB is repaired by a a is cell carrying the with CtIP, and the was used as a The of was significantly in with the These suggest that CtIP is important for efficient DSB repair through the HR the complex formation of BRCA1·CtIP·MRN is important for DSB repair, we of the CtIP at a critical phosphorylation and in BRCA1 (26Yu X. Chen J. Mol. Cell. Biol. 2004; 24: 9478-9486Crossref PubMed Scopus (312) Google Scholar). with CtIP when CtIP is by Because the interaction of BRCA1 with CtIP is required for efficient association of BRCA1 with MRN and suggest that the complex formation of BRCA1·CtIP·MRN is critical for DSB repair, and plays an important role in through the complex CtIP and the C of BRCA1 for ssDNA the of CtIP, is required for and resection of DSB to generate ssDNA C. Y. R.A. C. P. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In cells, CtIP is required for phosphorylation that on ssDNA as activation (26Yu X. Chen J. Mol. Cell. Biol. 2004; 24: 9478-9486Crossref PubMed Scopus (312) Google Scholar). CtIP is in a complex with MRN that carries DSB resection (16Jazayeri A. Falck J. Lukas C. Bartek J. Smith G.C. Lukas J. Jackson S.P. Nat. Cell Biol. 2006; 8: 37-45Crossref PubMed Scopus (882) Google Scholar, 18Myers J.S. Cortez D. J. Biol. Chem. 2006; 281: 9346-9350Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). CtIP plays a role in DSBs as in we IR-induced formation requires CtIP directly to ssDNA and been used as a for ssDNA formation (16Jazayeri A. Falck J. Lukas C. Bartek J. Smith G.C. Lukas J. Jackson S.P. Nat. Cell Biol. 2006; 8: 37-45Crossref PubMed Scopus (882) Google Scholar, 18Myers J.S. Cortez D. J. Biol. Chem. 2006; 281: 9346-9350Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, L. Elledge S.J. Science. 2003; PubMed Scopus Google Scholar). IR-induced in cells, that DSB resection is in S and G2 phases of the cell cycle (16Jazayeri A. Falck J. Lukas C. Bartek J. Smith G.C. Lukas J. Jackson S.P. Nat. Cell Biol. 2006; 8: 37-45Crossref PubMed Scopus (882) Google Scholar). of CtIP significantly reduced formation with the Lukas C. J. M. S. Bartek J. Baer R. Lukas J. Jackson S.P. Nature. PubMed Scopus Google Scholar). These suggest that CtIP is in DSB to that are required for Because BRCA1 is also required for activation and ssDNA formation S. M. L.C. Nat. Genet. 2002; PubMed Scopus Google Scholar, R. Cancer Res. 2006; PubMed Scopus Google Scholar), we its interaction with CtIP and MRN are important for IR-induced ssDNA formation in and its with Significantly, IR-induced formation is in with the BRCA1 cell line Because the interaction of BRCA1 and MRN is in the that a complex of BRCA1·CtIP·MRN is important for facilitating DNA resection at DSB to generate ssDNA and is required for efficient checkpoint activation and HR-mediated DSB repair. IR-induced of CtIP and MRN of to DSBs on Mre11 C. Y. R.A. C. P. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In cells, IR-induced CtIP are with that of Mre11 and However, the formation of CtIP and MRN to be CtIP not Mre11 and formation CtIP forms of IR-induced in cell line and its with These suggest that BRCA1, CtIP, and MRN a complex that is by DNA can be to DSBs in was Lukas C. J. M. S. Bartek J. Baer R. Lukas J. Jackson S.P. Nature. PubMed Scopus Google Scholar) with that CtIP interacts with MRN and DNA end In we that BRCA1 forms a complex with CtIP and MRN in a cell manner. studies suggest that not CtIP and MRN, but also formation of the complex BRCA1·CtIP·MRN are important for DSB end resection and HR-mediated DSB repair. These studies a critical mechanism for BRCA1 to in repair. that BRCA1, CtIP, and MRN a complex when the S and G2 phases of the cell and the complex formation requires activity. CtIP directly interacts with in and the in vivo interaction of BRCA1 with MRN is largely dependent on the of been that the interaction of BRCA1 and CtIP requires the phosphorylation at Ser327 (26Yu X. Chen J. Mol. Cell. Biol. 2004; 24: 9478-9486Crossref PubMed Scopus (312) Google Scholar). Here we that the BRCT required for CtIP are also needed for efficient interaction of BRCA1 with MRN. on we the that MRN is by CtIP to a complex with BRCA1 CtIP is by at at Ser327 in late S and G2 phases of the cell cycle. we that the N terminus of BRCA1 is also important for BRCA1 to with MRN in MRN is by CtIP to BRCA1 in S and complex formation of BRCA1·CtIP·MRN requires both the N terminus and C terminus of BRCA1 Significantly, studies that is not needed for BRCA1 to with MRN the cell cycle but is also important for association of BRCA1 with MRN. important in the of HR-mediated DSB repair in a cell cycle-dependent been that checkpoint to the of the interaction of BRCA1 with MRN (19Greenberg R.A. Sobhian B. Pathania S. Cantor S.B. Nakatani Y. Livingston D.M. Genes Dev. 2006; 20: 34-46Crossref PubMed Scopus (258) Google Scholar). propose that phosphorylation the complex formation of whereas phosphorylation on of the complex the interaction and to complex formation BRCA1 interacts with CtIP is by at MRN to a complex with BRCA1, and cell complex formation of BRCA1, CtIP, and MRN is a for of the the of the complex in to DNA with the by Lukas C. J. M. S. Bartek J. Baer R. Lukas J. Jackson S.P. Nature. PubMed Scopus Google Scholar), we that CtIP is important for DSB resection to generate ssDNA and HR-mediated DSB repair. CtIP is not in end resection for DSBs in S Lukas C. J. M. S. Bartek J. Baer R. Lukas J. Jackson S.P. Nature. PubMed Scopus Google Scholar) but is also needed when are with when DSBs are in studies also that the BRCA1 in CtIP and MRN association are significantly in ssDNA formation at the phosphorylation of CtIP at a critical to the association of BRCA1 with CtIP and the complex formation of is important for cellular Because the association of BRCA1 with CtIP, but not the interaction of CtIP with MRN, Chen and X. studies suggest a critical role of BRCA1 for HR-mediated DSB repair through its interaction with CtIP and MRN. been that CtIP the nuclease of Mre11 Lukas C. J. M. S. Bartek J. Baer R. Lukas J. Jackson S.P. Nature. PubMed Scopus Google Scholar). be to the association of BRCA1 with the nuclease of facilitating ssDNA formation at HR-mediated DSB repair is when the S and G2 phases of the cell and is important for (28Sonoda E. Hochegger H. Saberi A. Taniguchi Y. Takeda S. DNA Repair. 2006; 5: 1021-1029Crossref PubMed Scopus (386) Google Scholar). However, the mechanisms and the that are directly in DSB repair are not clear. studies a critical role for the complex formation of BRCA1·CtIP·MRN in DSB to generate evidence of BRCA1 participates in HR-mediated DSB repair. Because the complex is in S and G2 phases and requires studies suggest that one important mechanism by which participates in the of HR-mediated DSB repair is to complex formation of and for and for