Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.
References
[1]
Aguilera A, Gomez-Gonzalez B (2008) Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet 9: 204–217. doi: 10.1038/nrg2268. pmid:18227811
[2]
Allen C, Ashley AK, Hromas R, Nickoloff JA (2011) More forks on the road to replication stress recovery. J Mol Cell Biol 3: 4–12. doi: 10.1093/jmcb/mjq049. pmid:21278446
[3]
Petermann E, Helleday T (2010) Pathways of mammalian replication fork restart. Nat Rev Mol Cell Biol 11: 683–687. doi: 10.1038/nrm2974. pmid:20842177
[4]
Carr AM, Lambert S (2013) Replication stress-induced genome instability: the dark side of replication maintenance by homologous recombination. J Mol Biol 425: 4733–4744. doi: 10.1016/j.jmb.2013.04.023. pmid:23643490
[5]
Heller RC, Marians KJ (2006) Replisome assembly and the direct restart of stalled replication forks. Nat Rev Mol Cell Biol 7: 932–943. pmid:17139333 doi: 10.1038/nrm2058
[6]
Zeman MK, Cimprich KA (2014) Causes and consequences of replication stress. Nat Cell Biol 16: 2–9. doi: 10.1038/ncb2897. pmid:24366029
[7]
West SC (1997) Processing of recombination intermediates by the RuvABC proteins. Annu Rev Genet 31: 213–244. pmid:9442895 doi: 10.1146/annurev.genet.31.1.213
[8]
Yeeles JT, Poli J, Marians KJ, Pasero P (2013) Rescuing stalled or damaged replication forks. Cold Spring Harb Perspect Biol 5: a012815. doi: 10.1101/cshperspect.a012815. pmid:23637285
[9]
Costes A, Lambert SA (2012) Homologous recombination as a replication fork escort: fork-protection and recovery. Biomolecules 3: 39–71. doi: 10.3390/biom3010039. pmid:24970156
[10]
Arnaudeau C, Lundin C, Helleday T (2001) DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 307: 1235–1245. pmid:11292338 doi: 10.1006/jmbi.2001.4564
[11]
Hanada K, Budzowska M, Davies SL, van Drunen E, Onizawa H, et al. (2007) The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat Struct Mol Biol 14: 1096–1104. pmid:17934473 doi: 10.1038/nsmb1313
[12]
Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T (2010) Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol Cell 37: 492–502. doi: 10.1016/j.molcel.2010.01.021. pmid:20188668
[13]
Rass U (2013) Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes. Chromosoma 122: 499–515. doi: 10.1007/s00412-013-0431-z. pmid:24008669
[14]
Schwartz EK, Heyer WD (2011) Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes. Chromosoma 120: 109–127. doi: 10.1007/s00412-010-0304-7. pmid:21369956
[15]
Tay YD, Wu L (2010) Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing. J Biol Chem 285: 11427–11432. doi: 10.1074/jbc.M110.108399. pmid:20178992
[16]
Chapman JR, Taylor MR, Boulton SJ (2012) Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 47: 497–510. doi: 10.1016/j.molcel.2012.07.029. pmid:22920291
[17]
Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45: 247–271. doi: 10.1146/annurev-genet-110410-132435. pmid:21910633
[18]
Kakarougkas A, Jeggo PA (2014) DNA DSB repair pathway choice: an orchestrated handover mechanism. Br J Radiol 87: 20130685. doi: 10.1259/bjr.20130685. pmid:24363387
[19]
Truong LN, Li Y, Shi LZ, Hwang PY, He J, et al. (2013) Microhomology-mediated end joining and homologous recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells. Proc Natl Acad Sci USA 110: 7720–7725. doi: 10.1073/pnas.1213431110. pmid:23610439
[20]
Thangavel S, Berti M, Levikova M, Pinto C, Gomathinayagam S, et al. (2015) DNA2 drives processing and restart of reversed replication forks in human cells. J Cell Biol 208: 545–562. doi: 10.1083/jcb.201406100. pmid:25733713
[21]
Bouwman P, Aly A, Escandell JM, Pieterse M, Bartkova J, et al. (2010) 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat Struct Mol Biol 17: 688–695. doi: 10.1038/nsmb.1831. pmid:20453858
[22]
Bunting SF, Callen E, Wong N, Chen HT, Polato F, et al. (2010) 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141: 243–254. doi: 10.1016/j.cell.2010.03.012. pmid:20362325
[23]
Callen E, Di Virgilio M, Kruhlak MJ, Nieto-Soler M, Wong N, et al. (2013) 53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions. Cell 153: 1266–1280. doi: 10.1016/j.cell.2013.05.023. pmid:23727112
[24]
Escribano-Diaz C, Orthwein A, Fradet-Turcotte A, Xing M, Young JT, et al. (2013) A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol Cell 49: 872–883. doi: 10.1016/j.molcel.2013.01.001. pmid:23333306
[25]
Feng L, Fong KW, Wang J, Wang W, Chen J (2013) RIF1 counteracts BRCA1-mediated end resection during DNA repair. J Biol Chem 288: 11135–11143. doi: 10.1074/jbc.M113.457440. pmid:23486525
[26]
Zimmermann M, Lottersberger F, Buonomo SB, Sfeir A, de Lange T (2013) 53BP1 regulates DSB repair using Rif1 to control 5' end resection. Science 339: 700–704. doi: 10.1126/science.1231573. pmid:23306437
[27]
Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, et al. (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26: 125–132. doi: 10.1093/mutage/geq052. pmid:21164193
[28]
Nimonkar AV, Genschel J, Kinoshita E, Polaczek P, Campbell JL, et al. (2011) BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev 25: 350–362. doi: 10.1101/gad.2003811. pmid:21325134
[29]
Zhu Z, Chung WH, Shim EY, Lee SE, Ira G (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134: 981–994. doi: 10.1016/j.cell.2008.08.037. pmid:18805091
[30]
Chandramouly G, Kwok A, Huang B, Willis NA, Xie A, et al. (2013) BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids. Nat Commun 4: 2404. doi: 10.1038/ncomms3404. pmid:23994874
[31]
Nagaraju G, Hartlerode A, Kwok A, Chandramouly G, Scully R (2009) XRCC2 and XRCC3 regulate the balance between short- and long-tract gene conversions between sister chromatids. Mol Cell Biol 29: 4283–4294. doi: 10.1128/MCB.01406-08. pmid:19470754
[32]
Kim H-S, Chen Q, Kim S-K, Nickoloff JA, Hromas R, et al. (2014) The DDN catalytic motif is required for Metnase functions in NHEJ repair and replication restart. J Biol Chem 289: 10930–10938. doi: 10.1074/jbc.M113.533216. pmid:24573677
[33]
Clements PM, Breslin C, Deeks ED, Byrd PJ, Ju L, et al. (2004) The ataxia-oculomotor apraxia 1 gene product has a role distinct from ATM and interacts with the DNA strand break repair proteins XRCC1 and XRCC4. DNA Repair 3: 1493–1502. pmid:15380105 doi: 10.1016/j.dnarep.2004.06.017
[34]
Shiloh Y, Kastan MB (2001) ATM: genome stability, neuronal development, and cancer cross paths. Adv Cancer Res 83: 209–254. pmid:11665719 doi: 10.1016/s0065-230x(01)83007-4
[35]
De Haro LP, Wray J, Williamson EA, Durant ST, Corwin L, et al. (2010) Metnase promotes restart and repair of stalled and collapsed replication forks. Nucleic Acids Res 38: 5681–5691. doi: 10.1093/nar/gkq339. pmid:20457750
[36]
Hromas R, Williamson E, Fnu S, Lee Y-J, Park S-J, et al. (2012) Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart. Oncogene 31: 4245–4254. doi: 10.1038/onc.2011.586. pmid:22231448
[37]
Bennardo N, Cheng A, Huang N, Stark JM (2008) Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet 4: e1000110. doi: 10.1371/journal.pgen.1000110. pmid:18584027
[38]
Bennardo N, Stark JM (2010) ATM limits incorrect end utilization during non-homologous end joining of multiple chromosome breaks. PLoS Genet 6: e1001194. doi: 10.1371/journal.pgen.1001194. pmid:21079684
[39]
Lu H, Guo X, Meng X, Liu J, Wray J, et al. (2005) The BRCA2-interacting protein BCCIP functions in RAD51 and BRCA2 focus formation and homologous recombinational repair. Mol Cell Biol 25: 1949–1957. pmid:15713648 doi: 10.1128/mcb.25.5.1949-1957.2005
[40]
Brenneman MA, Wagener BM, Miller CA, Allen C, Nickoloff JA (2002) XRCC3 controls the fidelity of homologous recombination: roles for XRCC3 in late stages of recombination. Mol Cell 10: 387–395. pmid:12191483 doi: 10.1016/s1097-2765(02)00595-6
[41]
Nagaraju G, Odate S, Xie A, Scully R (2006) Differential regulation of short- and long-tract gene conversion between sister chromatids by Rad51C. Mol Cell Biol 26: 8075–8086. pmid:16954385 doi: 10.1128/mcb.01235-06
[42]
Lo Y-C, Paffett KS, Amit O, Clikeman JA, Sterk R, et al. (2006) Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity. Mol Cell Biol 26: 4086–4094. pmid:16705162 doi: 10.1128/mcb.00136-06
[43]
Pohl T, Nickoloff JA (2008) Rad51-independent double-strand break repair by gene conversion requires Rad52 but not Rad55, Rad57, or Dmc1. Mol Cell Biol 28: 897–906. pmid:18039855 doi: 10.1128/mcb.00524-07
[44]
Tsukuda T, Lo YC, Krishna S, Sterk R, Osley MA, et al. (2009) INO80-dependent chromatin remodeling regulates early and late stages of mitotic homologous recombination. DNA Repair 8: 360–369. doi: 10.1016/j.dnarep.2008.11.014. pmid:19095087
[45]
Tauchi H, Kobayashi J, Morishima K, van Gent DC, Shiraishi T, et al. (2002) Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420: 93–98. pmid:12422221 doi: 10.1038/nature01125
[46]
Saleh-Gohari N, Helleday T (2004) Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells. Oncogene 23: 9136–9141. pmid:15480413 doi: 10.1038/sj.onc.1208178
[47]
Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, et al. (2005) Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434: 913–917. pmid:15829966 doi: 10.1038/nature03443
[48]
Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, et al. (2005) Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434: 917–921. pmid:15829967 doi: 10.1038/nature03445
[49]
Rehman FL, Lord CJ, Ashworth A (2010) Synthetic lethal approaches to breast cancer therapy. Nat Rev Clin Oncol 7: 718–724. doi: 10.1038/nrclinonc.2010.172. pmid:20956981
[50]
Nicolette ML, Lee K, Guo Z, Rani M, Chow JM, et al. (2010) Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA double-strand breaks. Nat Struct Mol Biol 17: 1478–1485. doi: 10.1038/nsmb.1957. pmid:21102445
[51]
Shao Z, Davis AJ, Fattah KR, So S, Sun J, et al. (2012) Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination. DNA Repair 11: 310–316. doi: 10.1016/j.dnarep.2011.12.007. pmid:22265216
[52]
Fnu S, Williamson EA, De Haro LP, Brenneman M, Wray J, et al. (2011) Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining. Proc Natl Acad Sci USA 108: 540–545. doi: 10.1073/pnas.1013571108. pmid:21187428
[53]
Zhou Y, Caron P, Legube G, Paull TT (2014) Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res 42: e19. doi: 10.1093/nar/gkt1309. pmid:24362840
[54]
Byun TS, Pacek M, Yee MC, Walter JC, Cimprich KA (2005) Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev 19: 1040–1052. pmid:15833913 doi: 10.1101/gad.1301205
[55]
Choi JH, Lindsey-Boltz LA, Kemp M, Mason AC, Wold MS, et al. (2010) Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling. Proc Natl Acad Sci USA 107: 13660–13665. doi: 10.1073/pnas.1007856107. pmid:20616048
[56]
Liu S, Opiyo SO, Manthey K, Glanzer JG, Ashley AK, et al. (2012) Distinct roles for DNA-PK, ATM, and ATR in RPA phosphorylation and checkpoint activation in response to replication stress. Nucleic Acids Res 40: 10780–10794. doi: 10.1093/nar/gks849. pmid:22977173
[57]
Smits VA, Warmerdam DO, Martin Y, Freire R (2010) Mechanisms of ATR-mediated checkpoint signalling. Front Biosci 15: 840–853. doi: 10.2741/3649
[58]
Roy R, Chun J, Powell SN (2012) BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer 12: 68–78. doi: 10.1038/nrc3181
[59]
Robison JG, Elliott J, Dixon K, Oakley GG (2004) Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks. J Biol Chem 279: 34802–34810. pmid:15180989 doi: 10.1074/jbc.m404750200
[60]
Takeda S, Nakamura K, Taniguchi Y, Paull TT (2007) Ctp1/CtIP and the MRN complex collaborate in the initial steps of homologous recombination. Mol Cell 28: 351–352. pmid:17996697 doi: 10.1016/j.molcel.2007.10.016
[61]
Williams RS, Williams JS, Tainer JA (2007) Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin template. Biochem Cell Biol 85: 509–520. pmid:17713585 doi: 10.1139/o07-069
[62]
Feng Z, Zhang J (2012) A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest. Nucleic Acids Res 40: 726–738. doi: 10.1093/nar/gkr748. pmid:21954437
Lee B- I, Nguyen LH, Barsky D, Fernandes M, Wilson DM 3rd (2002) Molecular interactions of human Exo1 with DNA. Nucleic Acids Res 30: 942–949. pmid:11842105 doi: 10.1093/nar/30.4.942
[65]
Sirbu BM, Couch FB, Feigerle JT, Bhaskara S, Hiebert SW, et al. (2011) Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev 25: 1320–1327. doi: 10.1101/gad.2053211. pmid:21685366
[66]
Sirbu BM, Couch FB, Cortez D (2012) Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat Protoc 7: 594–605. doi: 10.1038/nprot.2012.010. pmid:22383038
[67]
Sarbajna S, Davies D, West SC (2014) Roles of SLX1-SLX4, MUS81-EME1, and GEN1 in avoiding genome instability and mitotic catastrophe. Genes Dev 28: 1124–1136. doi: 10.1101/gad.238303.114. pmid:24831703
Huang J, Liu S, Bellani MA, Thazhathveetil AK, Ling C, et al. (2013) The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks. Mol Cell 52: 434–446. doi: 10.1016/j.molcel.2013.09.021. pmid:24207054
[70]
Couch FB, Bansbach CE, Driscoll R, Luzwick JW, Glick GG, et al. (2013) ATR phosphorylates SMARCAL1 to prevent replication fork collapse. Genes Dev 27: 1610–1623. doi: 10.1101/gad.214080.113. pmid:23873943
[71]
Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, et al. (2001) The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412: 557–561. pmid:11484058 doi: 10.1038/35087613
[72]
Wang M, Wu W, Wu W, Rosidi B, Zhang L, et al. (2006) PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways. Nucleic Acids Res 34: 6170–6182. pmid:17088286 doi: 10.1093/nar/gkl840
[73]
Helleday T (2011) The underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings. Mol Oncol 5: 387–393. doi: 10.1016/j.molonc.2011.07.001. pmid:21821475
[74]
Mateos-Gomez PA, Gong F, Nair N, Miller KM, Lazzerini-Denchi E, et al. (2015) Mammalian polymerase theta promotes alternative NHEJ and suppresses recombination. Nature 518: 254–257. doi: 10.1038/nature14157. pmid:25642960
[75]
Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, et al. (2015) Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature 518: 258–262. doi: 10.1038/nature14184. pmid:25642963
[76]
Kent T, Chandramouly G, McDevitt SM, Ozdemir AY, Pomerantz RT (2015) Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ. Nat Struct Mol Biol 22: 230–237. doi: 10.1038/nsmb.2961. pmid:25643323
[77]
Tomimatsu N, Mukherjee B, Catherine Hardebeck M, Ilcheva M, Vanessa Camacho C, et al. (2014) Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice. Nat Commun 5: 3561. doi: 10.1038/ncomms4561. pmid:24705021
[78]
Makharashvili N, Tubbs AT, Yang SH, Wang H, Barton O, et al. (2014) Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. Mol Cell 54: 1022–1033. doi: 10.1016/j.molcel.2014.04.011. pmid:24837676
[79]
Stewart JA, Campbell JL, Bambara RA (2010) Dna2 is a structure-specific nuclease, with affinity for 5'-flap intermediates. Nucleic Acids Res 38: 920–930. doi: 10.1093/nar/gkp1055. pmid:19934252
[80]
Gaedcke J, Grade M, Jung K, Camps J, Jo P, et al. (2010) Mutated KRAS results in overexpression of DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase, in rectal carcinomas. Genes Chromosomes Cancer 49: 1024–1034. doi: 10.1002/gcc.20811. pmid:20725992
[81]
Lenz G, Wright G, Dave SS, Xiao W, Powell J, et al. (2008) Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 359: 2313–2323. doi: 10.1056/NEJMoa0802885. pmid:19038878
Wilson WH (2013) Treatment strategies for aggressive lymphomas: what works? Hematology Am Soc Hematol Educ Program 2013: 584–590. doi: 10.1182/asheducation-2013.1.584. pmid:24319235
[84]
Roman Y, Oshige M, Lee Y-J, Goodwin K, Georgiadis MM, et al. (2007) Biochemical characterization of a SET and transposase fusion protein, Metnase (SETMAR) for its DNA binding and DNA cleavage activity. Biochemistry 46: 11369–11376. pmid:17877369 doi: 10.1021/bi7005477
[85]
Taghian DG, Nickoloff JA (1997) Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells. Mol Cell Biol 17: 6386–6393. pmid:9343400 doi: 10.1128/mcb.17.11.6386
[86]
Shaffer LG, McGowan-Jordan J, Schmid M, editors (2013) ISCN 2013: An International System for Human Cytogenetic Nomenclature Basel: Karger. 140 p.
[87]
Pathak R, Sarma A, Sengupta B, Dey SK, Khuda-Bukhsh AR (2007) Response to high LET radiation 12C (LET, 295 keV/microm) in M5 cells, a radio resistant cell strain derived from Chinese hamster V79 cells. Int J Radiat Biol 83: 53–63. pmid:17357440 doi: 10.1080/09553000601085964
[88]
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175: 184–191. pmid:3345800 doi: 10.1016/0014-4827(88)90265-0
