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Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection

2022; Elsevier BV; Volume: 298; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2022.101937

ISSN

1083-351X

Autores

Aditya Mojumdar, Nancy Adam, Jennifer A. Cobb,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5′-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5′-resection mediated by the nuclease/helicase complex Dna2–Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11–Rad50–Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs. The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5′-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5′-resection mediated by the nuclease/helicase complex Dna2–Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11–Rad50–Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs. DNA double-strand breaks (DSBs) can be repaired by two central pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ mediates the direct ligation of DNA ends without the requirement for end processing, whereas HR requires 5′ end resection. Both 5′ resection and end-bridging are important for repair pathway choice and downstream outcomes. Once resection initiates, repair by canonical NHEJ is no longer an option. This key step is regulated by a network of proteins, including Nej1, which was first identified as a core NHEJ factor (1Wu D. Topper L.M. Wilson T.E. Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae.Genetics. 2008; 178: 1237-1249Crossref PubMed Scopus (99) Google Scholar, 2Palmbos P.L. Wu D. Daley J.M. Wilson T.E. Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 complex to a double-strand break requires interactions with Yku80 and the Xrs2 FHA domain.Genetics. 2008; 180: 1809-1819Crossref PubMed Scopus (48) Google Scholar, 3Chen X. Tomkinson A.E. Yeast Nej1 is a key participant in the initial end binding and final ligation steps of nonhomologous end joining.J. Biol. Chem. 2011; 286: 4931-4940Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 5Frank-Vaillant M. Marcand S. NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway.Genes Dev. 2001; 15: 3005-3012Crossref PubMed Scopus (134) Google Scholar, 6Valencia M. Bentele M. Vaze M.B. Herrmann G. Kraus E. Lee S.E. Schär P. Haber J.E. NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae.Nature. 2001; 414: 666-669Crossref PubMed Scopus (183) Google Scholar, 7Zhang Y. Hefferin M.L. Chen L. Shim E.Y. Tseng H.M. Kwon Y. Sung P. Lee S.E. Tomkinson A.E. Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination.Nat. Struct. Mol. Biol. 2007; 14: 639-646Crossref PubMed Scopus (107) Google Scholar, 8Mahaney B.L. Lees-Miller S.P. Cobb J.A. The C-terminus of Nej1 is critical for nuclear localization and non-homologous end-joining.DNA Repair (Amst.). 2014; 14: 9-16Crossref PubMed Scopus (13) Google Scholar, 9Sorenson K.S. Mahaney B.L. Lees-Miller S.P. Cobb J.A. The non-homologous end-joining factor Nej1 inhibits resection mediated by Dna2-Sgs1 nuclease-helicase at DNA double strand breaks.J. Biol. Chem. 2017; 292: 14576-14586Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). yKu70–80 (Ku) and Mre11–Rad50–Xrs2 (MRX) are the first complexes that localize to DSBs and both are important for recruiting Nej1 (1Wu D. Topper L.M. Wilson T.E. Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae.Genetics. 2008; 178: 1237-1249Crossref PubMed Scopus (99) Google Scholar, 2Palmbos P.L. Wu D. Daley J.M. Wilson T.E. Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 complex to a double-strand break requires interactions with Yku80 and the Xrs2 FHA domain.Genetics. 2008; 180: 1809-1819Crossref PubMed Scopus (48) Google Scholar, 3Chen X. Tomkinson A.E. Yeast Nej1 is a key participant in the initial end binding and final ligation steps of nonhomologous end joining.J. Biol. Chem. 2011; 286: 4931-4940Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Cells lacking NEJ1 are as defective in end-joining repair as ku70Δ and dnl4Δ (3Chen X. Tomkinson A.E. Yeast Nej1 is a key participant in the initial end binding and final ligation steps of nonhomologous end joining.J. Biol. Chem. 2011; 286: 4931-4940Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 5Frank-Vaillant M. Marcand S. NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway.Genes Dev. 2001; 15: 3005-3012Crossref PubMed Scopus (134) Google Scholar, 6Valencia M. Bentele M. Vaze M.B. Herrmann G. Kraus E. Lee S.E. Schär P. Haber J.E. NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae.Nature. 2001; 414: 666-669Crossref PubMed Scopus (183) Google Scholar). Moreover, Nej1 also contributes to Ku stability, which protects the DNA ends from nucleolytic degradation, and promotes Lif1-Dnl4–mediated ligation (2Palmbos P.L. Wu D. Daley J.M. Wilson T.E. Recruitment of Saccharomyces cerevisiae Dnl4-Lif1 complex to a double-strand break requires interactions with Yku80 and the Xrs2 FHA domain.Genetics. 2008; 180: 1809-1819Crossref PubMed Scopus (48) Google Scholar, 3Chen X. Tomkinson A.E. Yeast Nej1 is a key participant in the initial end binding and final ligation steps of nonhomologous end joining.J. Biol. Chem. 2011; 286: 4931-4940Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Zhang Y. Hefferin M.L. Chen L. Shim E.Y. Tseng H.M. Kwon Y. Sung P. Lee S.E. Tomkinson A.E. Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination.Nat. Struct. Mol. Biol. 2007; 14: 639-646Crossref PubMed Scopus (107) Google Scholar, 8Mahaney B.L. Lees-Miller S.P. Cobb J.A. The C-terminus of Nej1 is critical for nuclear localization and non-homologous end-joining.DNA Repair (Amst.). 2014; 14: 9-16Crossref PubMed Scopus (13) Google Scholar). Nej1 also functions in collaboration with MRX to bridge DNA ends at the DSB. The structural features of the MRX complex are critical for end-bridging, and deletion of NEJ1 results in end-bridging defects that are additive with rad50 mutants (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 10Hopfner K.P. Karcher A. Craig L. Woo T.T. Carney J.P. Tainer J.A. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.Cell. 2001; 105: 473-485Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 11Wiltzius J.J. Hohl M. Fleming J.C. Petrini J.H. The Rad50 hook domain is a critical determinant of Mre11 complex functions.Nat. Struct. Mol. Biol. 2005; 12: 403-407Crossref PubMed Scopus (117) Google Scholar, 12Hohl M. Kwon Y. Galván S.M. Xue X. Tous C. Aguilera A. Sung P. Petrini J.H. The Rad50 coiled-coil domain is indispensable for Mre11 complex functions.Nat. Struct. Mol. Biol. 2011; 18: 1124-1131Crossref PubMed Scopus (75) Google Scholar, 13Hohl M. Kochańczyk T. Tous C. Aguilera A. Krężel A. Petrini J.H. Interdependence of the rad50 hook and globular domain functions.Mol. Cell. 2015; 57: 479-491Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). While Nej1 and MRX both contribute to DNA end-bridging, Nej1 functions antagonistically to MRX as it inhibits 5′ DNA resection. Currently, few mechanistic details exist for how Nej1 inhibits resection, although previous work showed that Nej1 inhibits Dna2 interactions with Sgs1 and Mre11 (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). As work with Nej1 continues to emerge, it is becoming clear that its role in DSB repair involves more than stimulating Dnl4 ligase and stabilizing Ku during NHEJ. 5′ DNA resection occurs through a two-step process (14Symington L.S. Mechanism and regulation of DNA end resection in eukaryotes.Crit. Rev. Biochem. Mol. Biol. 2016; 51: 195-212Crossref PubMed Scopus (232) Google Scholar). First, Sae2, the yeast homolog of human CtIP, activates Mre11 endonuclease to initiate DNA resection, which also promotes Ku dissociation from the DNA ends (15Cannavo E. Cejka P. Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks.Nature. 2014; 514: 122-125Crossref PubMed Scopus (275) Google Scholar, 16Cejka P. DNA end resection: Nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Second, long-range resection follows, which is mediated by two functionally redundant 5′ to 3′ nucleases, Dna2, in complex with Sgs1, and Exo1 (16Cejka P. DNA end resection: Nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 17Zhu 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 (785) Google Scholar). Mre11 endonuclease activity is less critical for initiating 5′ resection than its physical presence at DSBs because both Exo1 and Dna2-Sgs1 can serve as compensatory back-ups, however both long-range nucleases require MRX for their localization (9Sorenson K.S. Mahaney B.L. Lees-Miller S.P. Cobb J.A. The non-homologous end-joining factor Nej1 inhibits resection mediated by Dna2-Sgs1 nuclease-helicase at DNA double strand breaks.J. Biol. Chem. 2017; 292: 14576-14586Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 17Zhu 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 (785) Google Scholar, 18Mimitou E.P. Symington L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.Nature. 2008; 455: 770-774Crossref PubMed Scopus (764) Google Scholar). Exo1 has high affinity for DNA ends and can initiate resection in mre11 nuclease dead (nd) mutants only when KU70 is deleted (19Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (173) Google Scholar, 20Mimitou E.P. Symington L.S. Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2.EMBO J. 2010; 29: 3358-3369Crossref PubMed Scopus (225) Google Scholar). By contrast, when NEJ1 is deleted, Exo1-mediated resection did not occur indicating that a certain level of Ku is maintained at DSBs in nej1Δ mutants (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 9Sorenson K.S. Mahaney B.L. Lees-Miller S.P. Cobb J.A. The non-homologous end-joining factor Nej1 inhibits resection mediated by Dna2-Sgs1 nuclease-helicase at DNA double strand breaks.J. Biol. Chem. 2017; 292: 14576-14586Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Regulation of Dna2-dependent resection seems to be more complex than Exo1, which appears to only require DNA ends not protected by Ku. Furthermore, understanding the function of Dna2 at DSBs has been challenging because DNA2 is an essential gene involved in Okazaki fragment processing and cannot be deleted (21Budd M.E. Campbell J.L. A new yeast gene required for DNA replication encodes a protein with homology to DNA helicases.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (150) Google Scholar, 22Budd M.E. Choe W.C. Campbell J.L. The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo.J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 23Jia P.P. Junaid M. Ma Y.B. Ahmad F. Jia Y.F. Li W.G. Pei D.S. Role of human DNA2 (hDNA2) as a potential target for cancer and other diseases: A systematic review.DNA Repair (Amst.). 2017; 59: 9-19Crossref PubMed Scopus (14) Google Scholar, 24Kumar S. Peng X. Daley J. Yang L. Shen J. Nguyen N. Bae G. Niu H. Peng Y. Hsieh H.J. Wang L. Rao C. Stephan C.C. Sung P. Ira G. et al.Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.Oncogenesis. 2017; 6e319Crossref Scopus (37) Google Scholar, 25Bae S.H. Bae K.H. Kim J.A. Seo Y.S. RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.Nature. 2001; 412: 456-461Crossref PubMed Scopus (283) Google Scholar). Earlier work showed that the lethality of dna2Δ can be suppressed by disruption of PIF1 helicase and that the frequency of 5′ resection decreased at a DSB in dna2Δ pif1-m2 mutants (19Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (173) Google Scholar, 26Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta.Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (166) Google Scholar). In the absence of Mre11 nuclease activity, resection initiates primarily through Dna2, independently of KU status (27Gobbini E. Villa M. Gnugnoli M. Menin L. Clerici M. Longhese M.P. Sae2 function at DNA double-strand breaks is bypassed by dampening Tel1 or Rad53 activity.PLoS Genet. 2015; 11e1005685Crossref PubMed Scopus (37) Google Scholar, 28Yu T.Y. Kimble M.T. Symington L.S. Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11961-E11969Crossref PubMed Scopus (25) Google Scholar, 29Arora S. Deshpande R.A. Budd M. Campbell J. Revere A. Zhang X. Schmidt K.H. Paull T.T. Genetic separation of Sae2 nuclease activity from Mre11 nuclease functions in budding yeast.Mol. Cell. Biol. 2017; 37e00156-17Crossref PubMed Scopus (8) Google Scholar). Moreover, using nuclease-deficient dna2-1 (P504→S), Dna2 and Mre11 showed functional redundancy for processing the ends of DSBs after radiation treatment (30Budd M.E. Campbell J.L. Interplay of Mre11 nuclease with Dna2 plus Sgs1 in Rad51-dependent recombinational repair.PLoS One. 2009; 4e4267Crossref PubMed Scopus (57) Google Scholar). Most work-describing Dna2 at DSBs has been performed in surrogate, by deleting SGS1 (16Cejka P. DNA end resection: Nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 31Bae S.H. Choi E. Lee K.H. Park J.S. Lee S.H. Seo Y.S. Dna2 of Saccharomyces cerevisiae possesses a single-stranded DNA-specific endonuclease activity that is able to act on double-stranded DNA in the presence of ATP.J. Biol. Chem. 1998; 273: 26880-26890Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, those studies cannot explain the greater IR and UV sensitivity of dna2-1 sgs1Δ mutants than single mutant counterparts (32Budd M.E. Campbell J.L. The pattern of sensitivity of yeast dna2 mutants to DNA damaging agents suggests a role in DSB and postreplication repair pathways.Mutat. Res. 2000; 459: 173-186Crossref PubMed Scopus (61) Google Scholar) and would not be able to identify any potential function(s) for Dna2 at DSBs independently of Sgs1. In humans, CtIP was shown to be another pathway for Dna2 recruitment to DSB (33Hoa N.N. Kobayashi J. Omura M. Hirakawa M. Yang S.H. Komatsu K. Paull T.T. Takeda S. Sasanuma H. BRCA1 and CtIP are both required to recruit Dna2 at double-strand breaks in homologous recombination.PLoS One. 2015; 10e0124495Crossref Scopus (26) Google Scholar). While this has yet to be demonstrated in yeast with Sae2, recently it was shown that Sae2 stimulates the nuclease and helicase activity of Dna2-Sgs1 in vitro (34Daley J.M. Jimenez-Sainz J. Wang W. Miller A.S. Xue X. Nguyen K.A. Jensen R.B. Sung P. Enhancement of BLM-DNA2-mediated long-range DNA end resection by CtIP.Cell Rep. 2017; 21: 324-332Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 35Ceppi I. Howard S.M. Kasaciunaite K. Pinto C. Anand R. Seidel R. Cejka P. CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 8859-8869Crossref PubMed Scopus (32) Google Scholar). Sae2 also has a role in DNA end-bridging at DSBs (36Ferrari M. Dibitetto D. De Gregorio G. Eapen V.V. Rawal C.C. Lazzaro F. Tsabar M. Marini F. Haber J.E. Pellicioli A. Functional interplay between the 53BP1-ortholog Rad9 and the Mre11 complex regulates resection, end-tethering and repair of a double-strand break.PLoS Genet. 2015; 11e1004928Crossref PubMed Scopus (74) Google Scholar), a function conserved in humans and with Ctp1 in fission yeast (37Öz R. Howard S.M. Sharma R. Törnkvist H. Ceppi I. Kk S. Kristiansson E. Cejka P. Westerlund F. Phosphorylated CtIP bridges DNA to promote annealing of broken ends.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 21403-21412Crossref PubMed Scopus (11) Google Scholar, 38Andres S.N. Li Z.M. Erie D.A. Williams R.S. Ctp1 protein-DNA filaments promote DNA bridging and DNA double-strand break repair.J. Biol. Chem. 2019; 294: 3312-3320Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). As both Nej1 and Sae2 have roles in end-bridging, yet function antagonistically to inhibit and promote resection respectively, investigating their relationship at DSBs is needed. In the present work, we show that Sae2 at DSBs is a key factor in Dna2 recruitment. Nej1 binds and inhibits Sae2 interactions with each component of the MRX complex and its interaction with Dna2. We also demonstrate that Nej1 functions in opposition to Dna2 and Sae2 in DNA end processing at DSBs. The deletion of NEJ1 led to increased 5′ resection and Sae2-dependent recovery of Dna2 at the break. We also show that deletion of NEJ1 can suppress the synthetic lethality (SL) of sae2Δ sgs1Δ through a mechanism dependent on the nuclease activity of Dna2. By contrast, epistatic end-bridging defects were seen in cells harboring NEJ1 and SAE2 deletions. Thus, distinct from their opposing relationship in regulating 5′ resection, Nej1–Sae2 interactions might restrict the mobility DNA ends at the break, an event important for both NHEJ and HR repair at DSBs. Sae2 initiates DNA end-resection by activating Mre11 endonuclease (16Cejka P. DNA end resection: Nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). By contrast, Nej1 interacts with the C-terminus of Mre11 and inhibits resection (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 9Sorenson K.S. Mahaney B.L. Lees-Miller S.P. Cobb J.A. The non-homologous end-joining factor Nej1 inhibits resection mediated by Dna2-Sgs1 nuclease-helicase at DNA double strand breaks.J. Biol. Chem. 2017; 292: 14576-14586Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Because these factors regulate 5′ resection in opposition and both depend on MRX for their localization (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 28Yu T.Y. Kimble M.T. Symington L.S. Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E11961-E11969Crossref PubMed Scopus (25) Google Scholar), we were prompted to investigate the interplay between them at the site-specific homothallic (HO)-DSB. First, we performed chromatin immuno-precipitation (ChIP) on Sae2 with primers located 0.6 kb from the DSB (Fig. 1A). Consistent with previous work, Sae2 decreased to background levels in mre11Δ mutants (Fig. 1B). By contrast, Sae2 recovery increased ∼2-fold in nej1Δ mutants from 40 min to 3 h after HO induction (Fig. 1, B and C). This was not an indirect consequence of disrupting NHEJ repair in general because Sae2 did not increase in cells where KU70 or DNL4 was deleted (Fig. 1B). Next, we assessed the importance of Sae2 in Nej1 localization. No change was seen in Nej1 recovery in sae2Δ mutants, which was somewhat surprising given that Ku70 recovery increased in sae2Δ mutants (Fig. S1A, (19Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (173) Google Scholar)), and Nej1 recovery in mre11Δ was reduced to background (Fig. 1D). To determine whether there was a physical interaction between Nej1 and Sae2, we next performed yeast two-hybrid (Y2H) as previously described (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 8Mahaney B.L. Lees-Miller S.P. Cobb J.A. The C-terminus of Nej1 is critical for nuclear localization and non-homologous end-joining.DNA Repair (Amst.). 2014; 14: 9-16Crossref PubMed Scopus (13) Google Scholar). This approach was used because Nej1 has a short half-life, making coimmunoprecipitation methods difficult (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 5Frank-Vaillant M. Marcand S. NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway.Genes Dev. 2001; 15: 3005-3012Crossref PubMed Scopus (134) Google Scholar, 8Mahaney B.L. Lees-Miller S.P. Cobb J.A. The C-terminus of Nej1 is critical for nuclear localization and non-homologous end-joining.DNA Repair (Amst.). 2014; 14: 9-16Crossref PubMed Scopus (13) Google Scholar, 39Carter S.D. Vigasová D. Chen J. Chovanec M. Aström S.U. Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 12037-12042Crossref PubMed Scopus (15) Google Scholar, 40Deshpande R.A. Wilson T.E. Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4.DNA Repair (Amst.). 2007; 6: 1507-1516Crossref PubMed Scopus (47) Google Scholar). Sae2 was expressed as hemagglutinin (HA)-tagged prey and Nej1 was expressed as LexA-tagged bait (4Mojumdar A. Sorenson K. Hohl M. Toulouze M. Lees-Miller S.P. Dubrana K. Cobb J.A. Nej1 interacts with Mre11 to regulate tethering and Dna2 binding at DNA double-strand breaks.Cell Rep. 2019; 28: 1564-1573.e3Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 8Mahaney B.L. Lees-Miller S.P. Cobb J.A. 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Functional interplay between the 53BP1-ortholog Rad9 and the Mre11 complex regulates resection, end-tethering and repair of a double-strand break.PLoS Genet.

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