The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.

Institute of Molecular Cancer Research University of Zurich 8057 Zurich Switzerland and

Institute of Molecular Cancer Research University of Zurich 8057 Zurich Switzerland and; Institute of Molecular Genetics Academy of Sciences of the Czech Republic 14300 Prague Czech Republic

Institute of Molecular Genetics Academy of Sciences of the Czech Republic 14300 Prague Czech Republic

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Jackson S. P., Bartek J. (2009) The DNA-damage response in human biology and disease. Nature 461, 1071–1078 PubMed PMC

Khanna K. K., Jackson S. P. (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat. Genet. 27, 247–254 PubMed

Longhese M. P., Bonetti D., Guerini I., Manfrini N., Clerici M. (2009) DNA double-strand breaks in meiosis: checking their formation, processing and repair. DNA Repair 8, 1127–1138 PubMed

Soulas-Sprauel P., Rivera-Munoz P., Malivert L., Le Guyader G., Abramowski V., Revy P., de Villartay J. P. (2007) V(D)J and immunoglobulin class switch recombinations: a paradigm to study the regulation of DNA end-joining. Oncogene 26, 7780–7791 PubMed

Lieber M. R. (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211 PubMed PMC

San Filippo J., Sung P., Klein H. (2008) Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257 PubMed

Heyer W. D., Ehmsen K. T., Liu J. (2010) Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–139 PubMed PMC

Mimitou E. P., Symington L. S. (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 PubMed PMC

Zhu Z., Chung W. H., Shim E. Y., Lee S. E., Ira G. (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134, 981–994 PubMed PMC

Cejka P., Cannavo E., Polaczek P., Masuda-Sasa T., Pokharel S., Campbell J. L., Kowalczykowski S. C. (2010) DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467, 112–116 PubMed PMC

Nicolette M. L., Lee K., Guo Z., Rani M., Chow J. M., Lee S. E., Paull T. T. (2010) Mre11-Rad50-Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nat. Struct. Mol. Biol. 17, 1478–1485 PubMed PMC

Shim E. Y., Chung W. H., Nicolette M. L., Zhang Y., Davis M., Zhu Z., Paull T. T., Ira G., Lee S. E. (2010) Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks. EMBO J. 29, 3370–3380 PubMed PMC

Cannavo E., Cejka P., Kowalczykowski S. C. (2013) Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection. Proc. Natl. Acad. Sci. U.S.A. 110, E1661–E1668 PubMed PMC

Niu H., Chung W. H., Zhu Z., Kwon Y., Zhao W., Chi P., Prakash R., Seong C., Liu D., Lu L., Ira G., Sung P. (2010) Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature 467, 108–111 PubMed PMC

Truong L. N., Li Y., Shi L. Z., Hwang P. Y., He J., Wang H., Razavian N., Berns M. W., Wu X. (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. U.S.A. 110, 7720–7725 PubMed PMC

Gravel S., Chapman J. R., Magill C., Jackson S. P. (2008) DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev. 22, 2767–2772 PubMed PMC

Nimonkar A. V., Genschel J., Kinoshita E., Polaczek P., Campbell J. L., Wyman C., Modrich P., Kowalczykowski S. C. (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 PubMed PMC

Karanja K. K., Cox S. W., Duxin J. P., Stewart S. A., Campbell J. L. (2012) DNA2 and EXO1 in replication-coupled, homology-directed repair and in the interplay between HDR and the FA/BRCA network. Cell Cycle 11, 3983–3996 PubMed PMC

Shibata A., Moiani D., Arvai A. S., Perry J., Harding S. M., Genois M. M., Maity R., van Rossum-Fikkert S., Kertokalio A., Romoli F., Ismail A., Ismalaj E., Petricci E., Neale M. J., Bristow R. G., Masson J. Y., Wyman C., Jeggo P. A., Tainer J. A. (2014) DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. Mol. Cell 53, 7–18 PubMed PMC

Bernstein K. A., Gangloff S., Rothstein R. (2010) The RecQ DNA helicases in DNA repair. Annu. Rev. Genet. 44, 393–417 PubMed PMC

Yan H., McCane J., Toczylowski T., Chen C. (2005) Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair. J. Cell Biol. 171, 217–227 PubMed PMC

Liao S., Toczylowski T., Yan H. (2008) Identification of the Xenopus DNA2 protein as a major nuclease for the 5′→3′ strand-specific processing of DNA ends. Nucleic Acids Res. 36, 6091–6100 PubMed PMC

Liao S., Guay C., Toczylowski T., Yan H. (2012) Analysis of MRE11's function in the 5′→3′ processing of DNA double-strand breaks. Nucleic Acids Res. 40, 4496–4506 PubMed PMC

Saydam N., Kanagaraj R., Dietschy T., Garcia P. L., Peña-Diaz J., Shevelev I., Stagljar I., Janscak P. (2007) Physical and functional interactions between Werner syndrome helicase and mismatch-repair initiation factors. Nucleic Acids Res. 35, 5706–5716 PubMed PMC

Adams K. E., Medhurst A. L., Dart D. A., Lakin N. D. (2006) Recruitment of ATR to sites of ionising radiation-induced DNA damage requires ATM and components of the MRN protein complex. Oncogene 25, 3894–3904 PubMed PMC

Sartori A. A., Lukas C., Coates J., Mistrik M., Fu S., Bartek J., Baer R., Lukas J., Jackson S. P. (2007) Human CtIP promotes DNA end resection. Nature 450, 509–514 PubMed PMC

Yang J., O'Donnell L., Durocher D., Brown G. W. (2012) RMI1 promotes DNA replication fork progression and recovery from replication fork stress. Mol. Cell Biol. 32, 3054–3064 PubMed PMC

Orren D. K., Brosh R. M., Jr., Nehlin J. O., Machwe A., Gray M. D., Bohr V. A. (1999) Enzymatic and DNA binding properties of purified WRN protein: high affinity binding to single-stranded DNA but not to DNA damage induced by 4NQO. Nucleic Acids Res. 27, 3557–3566 PubMed PMC

Kanagaraj R., Saydam N., Garcia P. L., Zheng L., Janscak P. (2006) Human RECQ5β helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork. Nucleic Acids Res. 34, 5217–5231 PubMed PMC

El-Shemerly M., Janscak P., Hess D., Jiricny J., Ferrari S. (2005) Degradation of human exonuclease 1b upon DNA synthesis inhibition. Cancer Res. 65, 3604–3609 PubMed

Henricksen L. A., Umbricht C. B., Wold M. S. (1994) Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 PubMed

Masuda-Sasa T., Imamura O., Campbell J. L. (2006) Biochemical analysis of human Dna2. Nucleic Acids Res. 34, 1865–1875 PubMed PMC

Pfaffl M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45. PubMed PMC

Bennardo N., Cheng A., Huang N., Stark J. M. (2008) Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110. PubMed PMC

Gunn A., Stark J. M. (2012) I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol. Biol. 920, 379–391 PubMed

Richardson C., Moynahan M. E., Jasin M. (1998) Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev. 12, 3831–3842 PubMed PMC

Karow J. K., Chakraverty R. K., Hickson I. D. (1997) The Bloom's syndrome gene product is a 3′-5′ DNA helicase. J. Biol. Chem. 272, 30611–30614 PubMed

Brosh R. M., Jr., Waheed J., Sommers J. A. (2002) Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase. J. Biol. Chem. 277, 23236–23245 PubMed

Shen J. C., Gray M. D., Oshima J., Kamath-Loeb A. S., Fry M., Loeb L. A. (1998) Werner syndrome protein: I: DNA helicase and DNA exonuclease reside on the same polypeptide. J. Biol. Chem. 273, 34139–34144 PubMed

Kamath-Loeb A. S., Shen J. C., Loeb L. A., Fry M. (1998) Werner syndrome protein: II: characterization of the integral 3′ → 5′ DNA exonuclease. J. Biol. Chem. 273, 34145–34150 PubMed

Kim J. H., Kim H. D., Ryu G. H., Kim D. H., Hurwitz J., Seo Y. S. (2006) Isolation of human DNA2 endonuclease and characterization of its enzymatic properties. Nucleic Acids Res. 34, 1854–1864 PubMed PMC

Avemann K., Knippers R., Koller T., Sogo J. M. (1988) Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Mol. Cell Biol. 8, 3026–3034 PubMed PMC

Lee J. W., Harrigan J., Opresko P. L., Bohr V. A. (2005) Pathways and functions of the Werner syndrome protein. Mech. Ageing Dev. 126, 79–86 PubMed

Kanagaraj R., Parasuraman P., Mihaljevic B., van Loon B., Burdova K., König C., Furrer A., Bohr V. A., Hübscher U., Janscak P. (2012) Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage. Nucleic Acids Res. 40, 8449–8459 PubMed PMC

Stark J. M., Pierce A. J., Oh J., Pastink A., Jasin M. (2004) Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol. Cell Biol. 24, 9305–9316 PubMed PMC

Wu L., Hickson I. D. (2003) The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 PubMed

Yin J., Sobeck A., Xu C., Meetei A. R., Hoatlin M., Li L., Wang W. (2005) BLAP75, an essential component of Bloom's syndrome protein complexes that maintain genome integrity. EMBO J. 24, 1465–1476 PubMed PMC

Wu L., Bachrati C. Z., Ou J., Xu C., Yin J., Chang M., Wang W., Li L., Brown G. W., Hickson I. D. (2006) BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates. Proc. Natl. Acad. Sci. U.S.A. 103, 4068–4073 PubMed PMC

Xu D., Guo R., Sobeck A., Bachrati C. Z., Yang J., Enomoto T., Brown G. W., Hoatlin M. E., Hickson I. D., Wang W. (2008) RMI, a new OB-fold complex essential for Bloom syndrome protein to maintain genome stability. Genes Dev. 22, 2843–2855 PubMed PMC

Cheng W. H., von Kobbe C., Opresko P. L., Arthur L. M., Komatsu K., Seidman M. M., Carney J. P., Bohr V. A. (2004) Linkage between Werner syndrome protein and the Mre11 complex via Nbs1. J. Biol. Chem. 279, 21169–21176 PubMed

Lan L., Nakajima S., Komatsu K., Nussenzweig A., Shimamoto A., Oshima J., Yasui A. (2005) Accumulation of Werner protein at DNA double-strand breaks in human cells. J. Cell Sci. 118, 4153–4162 PubMed

Tomimatsu N., Mukherjee B., Deland K., Kurimasa A., Bolderson E., Khanna K. K., Burma S. (2012) Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions. DNA Repair 11, 441–448 PubMed PMC

Yu C. E., Oshima J., Fu Y. H., Wijsman E. M., Hisama F., Alisch R., Matthews S., Nakura J., Miki T., Ouais S., Martin G. M., Mulligan J., Schellenberg G. D. (1996) Positional cloning of the Werner's syndrome gene. Science 272, 258–262 PubMed

Salk D., Au K., Hoehn H., Martin G. M. (1981) Cytogenetics of Werner's syndrome cultured skin fibroblasts: variegated translocation mosaicism. Cytogenet. Cell Genet. 30, 92–107 PubMed

Fukuchi K., Martin G. M., Monnat R. J., Jr. (1989) Mutator phenotype of Werner syndrome is characterized by extensive deletions. Proc. Natl. Acad. Sci. U.S.A. 86, 5893–5897 PubMed PMC

Melcher R., von Golitschek R., Steinlein C., Schindler D., Neitzel H., Kainer K., Schmid M., Hoehn H. (2000) Spectral karyotyping of Werner syndrome fibroblast cultures. Cytogenet. Cell Genet. 91, 180–185 PubMed

Bunting S. F., Nussenzweig A. (2013) End-joining, translocations and cancer. Nat. Rev. Cancer 13, 443–454 PubMed PMC

Symington L. S., Gautier J. (2011) Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 PubMed

Chen L., Huang S., Lee L., Davalos A., Schiestl R. H., Campisi J., Oshima J. (2003) WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair. Aging Cell 2, 191–199 PubMed

Perry J. J., Yannone S. M., Holden L. G., Hitomi C., Asaithamby A., Han S., Cooper P. K., Chen D. J., Tainer J. A. (2006) WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 13, 414–422 PubMed

Saintigny Y., Makienko K., Swanson C., Emond M. J., Monnat R. J., Jr. (2002) Homologous recombination resolution defect in Werner syndrome. Mol. Cell Biol. 22, 6971–6978 PubMed PMC

Swanson C., Saintigny Y., Emond M. J., Monnat R. J., Jr. (2004) The Werner syndrome protein has separable recombination and survival functions. DNA Repair 3, 475–482 PubMed

Bussen W., Raynard S., Busygina V., Singh A. K., Sung P. (2007) Holliday junction processing activity of the BLM-Topo IIIα-BLAP75 complex. J. Biol. Chem. 282, 31484–31492 PubMed

Raynard S., Bussen W., Sung P. (2006) A double Holliday junction dissolvasome comprising BLM, topoisomerase IIIα, and BLAP75. J. Biol. Chem. 281, 13861–13864 PubMed

Xue X., Raynard S., Busygina V., Singh A. K., Sung P. (2013) Role of replication protein A in double Holliday junction dissolution mediated by the BLM-Topo IIIα-RMI1-RMI2 protein complex. J. Biol. Chem. 288, 14221–14227 PubMed PMC