Advertisement

Activation of Tel1ATM kinase requires Rad50 ATPase and long nucleosome-free DNA but no DNA ends

  • Sarem Hailemariam
    Affiliations
    From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Sandeep Kumar
    Footnotes
    Affiliations
    From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Peter M. Burgers
    Correspondence
    To whom correspondence should be addressed. Tel.:314-362-3772; Fax:314-362-7183
    Affiliations
    From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Author Footnotes
    1 Present address: Intellia Therapeutics, Cambridge, MA 02139.
    3 The abbreviations used are: DSBdouble-stranded DNA breakATMataxia telangiectasia–mutatedATRATM- and Rad3-relatedPIKKPI3K-related protein kinaseHEATHuntington, elongation factor 3, A subunit of protein phosphatase 2A, and TOR1ssDNAsingle-stranded DNAMRXMre11–Rad50–Xrs2MRNMre11–Rad50–Nbs1RPAreplication protein A.
Open AccessPublished:May 09, 2019DOI:https://doi.org/10.1074/jbc.RA119.008410
      In Saccharomyces cerevisiae, Tel1 protein kinase, the ortholog of human ataxia telangiectasia–mutated (ATM), is activated in response to DNA double-strand breaks. Biochemical studies with human ATM and genetic studies in yeast suggest that recruitment and activation of Tel1ATM depends on the heterotrimeric MRXMRN complex, composed of Mre11, Rad50, and Xrs2 (human Nbs1). However, the mechanism of activation of Tel1 by MRX remains unclear, as does the role of effector DNA. Here we demonstrate that dsDNA and MRX activate Tel1 synergistically. Although minimal activation was observed with 80-mer duplex DNA, the optimal effector for Tel1 activation is long, nucleosome-free DNA. However, there is no requirement for DNA double-stranded termini. The ATPase activity of Rad50 is critical for activation. In addition to DNA and Rad50, either Mre11 or Xrs2, but not both, is also required. Each of the three MRX subunits shows a physical association with Tel1. Our study provides a model of how the individual subunits of MRX and DNA regulate Tel1 kinase activity.

      Introduction

      Genome stability mechanisms are essential for the normal function of cells. Genomic integrity can be disrupted because of errors caused during DNA replication and by both endogenous and exogenous DNA-damaging agents. Double-strand breaks (DSBs)
      The abbreviations used are: DSB
      double-stranded DNA break
      ATM
      ataxia telangiectasia–mutated
      ATR
      ATM- and Rad3-related
      PIKK
      PI3K-related protein kinase
      HEAT
      Huntington, elongation factor 3, A subunit of protein phosphatase 2A, and TOR1
      ssDNA
      single-stranded DNA
      MRX
      Mre11–Rad50–Xrs2
      MRN
      Mre11–Rad50–Nbs1
      RPA
      replication protein A.
      are among the most cytotoxic and deleterious forms of DNA lesions. The efficient repair of DSBs is essential for genome integrity. DSBs can be formed by exogenous agents, but they can also occur during normal cellular processes, such as during meiosis, immunoglobulin gene rearrangement, and DNA replication. Defective DSB repair is associated with developmental and immunological disorders and promotes carcinogenesis in humans (
      • Lavin M.F.
      • Shiloh Y.
      The genetic defect in ataxia-telangiectasia.
      ). To avoid chromosomal alterations and rearrangements, cells must detect and repair DSBs timely and properly. Eukaryotes respond to DSBs by promptly initiating a multipronged DNA damage response, which involves the initiation of a cell cycle checkpoint and DNA repair. The cell cycle checkpoint is an intricate signal transduction pathway that involves DNA damage sensing, a slowing down or inhibition of cell cycle progression, and DNA repair (
      • Oh J.
      • Symington L.S.
      Role of the Mre11 complex in preserving genome integrity.
      ).
      The yeast Saccharomyces cerevisiae Tel1 and Mec1 protein kinases are orthologs of mammalian ataxia telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR), respectively. They are essential regulators of cell cycle checkpoint initiation in yeast (
      • Abraham R.T.
      Cell cycle checkpoint signaling through the ATM and ATR kinases.
      ,
      • Mallory J.C.
      • Petes T.D.
      Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase.
      ). Tel1 and Mec1 belong to a superfamily of protein kinases that share a common C-terminal phosphatidylinositol-3′-OH kinase–like kinase (PIKK) domain. All members of the PIKK family are very large proteins (>250 kDa), in which the C-terminal catalytic kinase domain makes up only 5–10% of the total sequence. The nonkinase regions of PIKKs are comprised largely of tandem HEAT (Huntington, elongation factor 3, A subunit of protein phosphatase 2A, and TOR1) repeats and show very little sequence homology even within subfamilies. Structural analyses have revealed that HEAT repeats form helical structures that form large curved superstructures (
      • Perry J.
      • Kleckner N.
      The ATRs, ATMs, and TORs are giant HEAT repeat proteins.
      ,
      • Sawicka M.
      • Wanrooij P.H.
      • Darbari V.C.
      • Tannous E.
      • Hailemariam S.
      • Bose D.
      • Makarova A.V.
      • Burgers P.M.
      • Zhang X.
      The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organization.
      • Shiloh Y.
      ATM and related protein kinases: safeguarding genome integrity.
      ). In addition to harboring the kinase domain, the C-terminal region of PIKKs is also very highly conserved (
      • Bosotti R.
      • Isacchi A.
      • Sonnhammer E.L.
      FAT: a novel domain in PIK-related kinases.
      ,
      • Ogi H.
      • Goto G.H.
      • Ghosh A.
      • Zencir S.
      • Henry E.
      • Sugimoto K.
      Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition.
      ). Recent structural studies of Schizosaccharomyces pombe Tel1 show two Tel1 molecules that interact through the kinase and C-terminal domain to form a dimer (Fig. 1A) (
      • Wang X.
      • Chu H.
      • Lv M.
      • Zhang Z.
      • Qiu S.
      • Liu H.
      • Shen X.
      • Wang W.
      • Cai G.
      Structure of the intact ATM/Tel1 kinase.
      ).
      Figure thumbnail gr1
      Figure 1Tel1 kinase and the MRX complex. A, Tel1 and ATM kinases exist as dimers. The structure shown is that of human ATM (PDB code 4JSV). Yellow, HEAT repeats; blue, kinase and kinase-associated domains. NTD, N-terminal domain. B, schematic of the MRX complex in the presence and absence of ATP. The model is based on structural analysis of archaeal MR complexes. ATP binding by Rad50 transforms MRX from an open to closed conformation (see text for details). C, 7% SDS-PAGE analysis of purified Tel1, MRX, and MRX subunits and Rad53kd. Staining was performed with Coomassie Blue. M, size markers.
      Tel1 (ATM) and Mec1 (ATR) respond to different forms of DNA damage. Although Mec1 is activated by ssDNA coated with the single-stranded binding factor RPA, Tel1 responds primarily to DSBs (
      • Sanchez Y.
      • Desany B.A.
      • Jones W.J.
      • Liu Q.
      • Wang B.
      • Elledge S.J.
      Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways.
      ,
      • Usui T.
      • Ogawa H.
      • Petrini J.H.
      A DNA damage response pathway controlled by Tel1 and the Mre11 complex.
      • Zou L.
      • Elledge S.J.
      Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
      ). However, they share an overlapping number of downstream phosphorylation targets and show partial redundancy in their checkpoint function (
      • Mallory J.C.
      • Petes T.D.
      Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase.
      ). One of the initial targets of Tel1 in S. cerevisiae is histone H2A. Phosphorylation of H2A marks the chromatin surrounding the DNA lesion and is important for recruitment and retention of DNA repair proteins (
      • Downs J.A.
      • Lowndes N.F.
      • Jackson S.P.
      A role for Saccharomyces cerevisiae histone H2A in DNA repair.
      ). Phosphorylated H2A is bound by the scaffold adaptor protein Rad9. The association of Rad9 with chromatin facilitates recruitment of the effector kinase Rad53, the budding yeast functional homolog of mammalian Chk1 and Chk2 kinases (
      • Hammet A.
      • Magill C.
      • Heierhorst J.
      • Jackson S.P.
      Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.
      ). Phosphorylation of Rad53 by Tel1, followed by further autophosphorylation of Rad53, results in full activation of Rad53 (
      • Usui T.
      • Foster S.S.
      • Petrini J.H.
      Maintenance of the DNA damage checkpoint requires DNA damage-induced mediator protein oligomerization.
      ). Rad53 has many downstream targets, some of which couple cell cycle arrest with damage repair (
      • Usui T.
      • Ogawa H.
      • Petrini J.H.
      A DNA damage response pathway controlled by Tel1 and the Mre11 complex.
      ,
      • Usui T.
      • Foster S.S.
      • Petrini J.H.
      Maintenance of the DNA damage checkpoint requires DNA damage-induced mediator protein oligomerization.
      ,
      • Lee S.J.
      • Schwartz M.F.
      • Duong J.K.
      • Stern D.F.
      Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling.
      ).
      Recruitment of Tel1 to sites of DNA damage depends on Xrs2 (human Nbs1) protein. Xrs2 is a subunit of the Mre11–Rad50–Xrs2 (MRX) complex (
      • Nakada D.
      • Matsumoto K.
      • Sugimoto K.
      ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism.
      ,
      • You Z.
      • Chahwan C.
      • Bailis J.
      • Hunter T.
      • Russell P.
      ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.
      ). The MRX (human MRN) complex is involved in recognition and processing of DSBs. Mre11 and Rad50 are highly conserved and are found in all forms of life. Xrs2/Nbs1 is only present in eukaryotes and is not well conserved at the primary sequence level (
      • Aravind L.
      • Walker D.R.
      • Koonin E.V.
      Conserved domains in DNA repair proteins and evolution of repair systems.
      ). MRX has several enzymatic and scaffolding properties and plays major roles in DNA break metabolism. It is a crucial player in all aspects of DSB processing: damage detection, cell cycle checkpoint initiation, and facilitating and catalyzing repair of the lesion (
      • Oh J.
      • Symington L.S.
      Role of the Mre11 complex in preserving genome integrity.
      ,
      • Adelman C.A.
      • Petrini J.H.
      Division of labor: DNA repair and the cell cycle specific functions of the Mre11 complex.
      ,
      • Lamarche B.J.
      • Orazio N.I.
      • Weitzman M.D.
      The MRN complex in double-strand break repair and telomere maintenance.
      ). Mre11 belongs to the λ phosphatase family of enzymes and has both endo- and exonuclease activities (
      • Paull T.T.
      • Gellert M.
      The 3′ to 5′ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks.
      ,
      • Usui T.
      • Ohta T.
      • Oshiumi H.
      • Tomizawa J.
      • Ogawa H.
      • Ogawa T.
      Complex formation and functional versatility of Mre11 of budding yeast in recombination.
      ). Studies of budding yeast Mre11 show that mutations ablating its nuclease activity cause severe meiotic defects but result in only mild IR sensitivity, suggesting that the Mre11 nuclease activity is largely dispensable for DSB repair (
      • Furuse M.
      • Nagase Y.
      • Tsubouchi H.
      • Murakami-Murofushi K.
      • Shibata T.
      • Ohta K.
      Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.
      ,
      • Symington L.S.
      • Gautier J.
      Double-strand break end resection and repair pathway choice.
      ). Rad50 belongs to the ABC transporter family of ATPases and shares structural features with structural maintenance of chromosome proteins (
      • Aravind L.
      • Walker D.R.
      • Koonin E.V.
      Conserved domains in DNA repair proteins and evolution of repair systems.
      ,
      • Hopfner K.P.
      • Karcher A.
      • Shin D.S.
      • Craig L.
      • Arthur L.M.
      • Carney J.P.
      • Tainer J.A.
      Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
      ,
      • Raymond W.E.
      • Kleckner N.
      RAD50 protein of S. cerevisiae exhibits ATP-dependent DNA binding.
      ). Its ATP-binding domains are split and located at the N- and C termini, separated by long coil-coiled domains (
      • Melby T.E.
      • Ciampaglio C.N.
      • Briscoe G.
      • Erickson H.P.
      The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge.
      ,
      • Walker J.E.
      • Saraste M.
      • Runswick M.J.
      • Gay N.J.
      Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.
      ) (Fig. 1B). Active ATPase sites are only formed when two Rad50 molecules dimerize in an ATP-dependent manner (
      • Hopfner K.P.
      • Karcher A.
      • Shin D.S.
      • Craig L.
      • Arthur L.M.
      • Carney J.P.
      • Tainer J.A.
      Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
      ). A conserved zinc hook at the apex of the coil-coiled region, distal from the ATPase domains, also mediates Rad50 dimerization (
      • Hopfner K.P.
      • Craig L.
      • Moncalian G.
      • Zinkel R.A.
      • Usui T.
      • Owen B.A.
      • Karcher A.
      • Henderson B.
      • Bodmer J.L.
      • McMurray C.T.
      • Carney J.P.
      • Petrini J.H.
      • Tainer J.A.
      The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair.
      ). The ATPase domains of Rad50, along with the nuclease and DNA binding regions of Mre11, make up the catalytic core of the MRX complex (
      • Lammens K.
      • Bemeleit D.J.
      • Möckel C.
      • Clausing E.
      • Schele A.
      • Hartung S.
      • Schiller C.B.
      • Lucas M.
      • Angermüller C.
      • Söding J.
      • Strässer K.
      • Hopfner K.P.
      The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair.
      ,
      • Möckel C.
      • Lammens K.
      • Schele A.
      • Hopfner K.P.
      ATP driven structural changes of the bacterial Mre11:Rad50 catalytic head complex.
      ). Upon ATP binding by Rad50, this catalytic core retains a closed conformation, whereas ATP hydrolysis releases the MRX complex from the DNA (
      • Hopfner K.P.
      • Karcher A.
      • Shin D.S.
      • Craig L.
      • Arthur L.M.
      • Carney J.P.
      • Tainer J.A.
      Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
      ,
      • Anderson D.E.
      • Trujillo K.M.
      • Sung P.
      • Erickson H.P.
      Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy.
      • de Jager M.
      • van Noort J.
      • van Gent D.C.
      • Dekker C.
      • Kanaar R.
      • Wyman C.
      Human Rad50/Mre11 is a flexible complex that can tether DNA ends.
      ,
      • Hopfner 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.
      • Moreno-Herrero F.
      • de Jager M.
      • Dekker N.H.
      • Kanaar R.
      • Wyman C.
      • Dekker C.
      Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA.
      ) (Fig. 1B).
      Critical studies of the mammalian ATM and the MRN complex have resulted in a mechanistic framework for checkpoint initiation to DSBs (
      • Paull T.T.
      20 Years of Mre11 biology: no end in sight.
      ). In an independent approach, extensive genetic studies of S. cerevisiae Tel1 and the MRX complex have given us a comprehensive understanding of where and how these factors function in various DNA metabolic pathways and which activities and/or domains are essential in each of these pathways (
      • Oh J.
      • Symington L.S.
      Role of the Mre11 complex in preserving genome integrity.
      ,
      • Adelman C.A.
      • Petrini J.H.
      Division of labor: DNA repair and the cell cycle specific functions of the Mre11 complex.
      ). However, until now, biochemical studies with purified yeast proteins have been lacking. In this study, we present biochemical data that illuminate how DNA and MRX orchestrate the activation of the Tel1 checkpoint kinase. We show that dsDNA is indispensable for stimulation of Tel1 kinase by MRX. A DNA length with a minimum of 80 bp is required for robust activation. Furthermore, DNA wrapped up into a nucleosome is ineffective. Surprisingly, dsDNA ends were not required for activation of Tel1 by MRX in vitro, even though, on small DNA effectors, a significant stimulatory function of DNA ends can be detected. Analysis of individual subunits and different heterodimeric pairs of MRX in the presence of DNA reveal that Rad50 is critical for activating Tel1. This stimulatory effect of Rad50 is absolutely dependent on its ATPase activity.

      Results

      DNA is required for MRX to fully stimulate Tel1

      Previously, we described the overproduction and purification of S. cerevisiae Tel1 from yeast and described an initial structural analysis (
      • Sawicka M.
      • Wanrooij P.H.
      • Darbari V.C.
      • Tannous E.
      • Hailemariam S.
      • Bose D.
      • Makarova A.V.
      • Burgers P.M.
      • Zhang X.
      The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organization.
      ). When Tel1 was examined by EM, the main species was that of a dimer. Cryo-EM studies of human ATM and S. pombe Tel1 show that these orthologs are also homodimeric in structure (
      • Wang X.
      • Chu H.
      • Lv M.
      • Zhang Z.
      • Qiu S.
      • Liu H.
      • Shen X.
      • Wang W.
      • Cai G.
      Structure of the intact ATM/Tel1 kinase.
      ,
      • Lau W.C.
      • Li Y.
      • Liu Z.
      • Gao Y.
      • Zhang Q.
      • Huen M.S.
      Structure of the human dimeric ATM kinase.
      ).
      To allow for a comprehensive biochemical analysis of the MRX complex in its stimulation of Tel1 kinase activity, we purified each subunit separately from yeast, as well as the heterodimeric Mre11–Xrs2 complex. However, these highly purified preparations, particularly the Mre11–Xrs2 preparation, consistently showed protein kinase activity in the absence of Tel1 addition. In part, the contaminating kinase activity could be assigned to Tel1 itself, which copurified with these subunits. Because each subunit of the MRX complex interacts with Tel1 (see below), this finding was not unexpected. However, even when the MRX subunits were purified from a tel1Δ strain, detectable kinase activity remained. Mass spectrometry analysis of the purified preparations identified Tda1 as the co-purifying kinase (data not shown). TDA1 shows genetic interactions with topoisomerase 1, suggesting that it may play a role in DNA metabolism (
      • Reid R.J.
      • González-Barrera S.
      • Sunjevaric I.
      • Alvaro D.
      • Ciccone S.
      • Wagner M.
      • Rothstein R.
      Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage.
      ). A potential role of Tda1 in MRX-dependent DNA metabolic processes remains to be investigated. When we purified the MRX subunits and subassemblies from a tel1Δ tda1Δ strain of yeast, essentially no contaminating protein kinase activity was observed (Fig. 2A, lane 2), and these preparations were used in all of our studies (Fig. 1C).
      Figure thumbnail gr2
      Figure 2MRX and DNA synergistically activate Tel1 kinase. A, standard kinase reactions contained 200 nm Rad53kd and 50 μm [γ-32P]ATP in kinase buffer with or without 30 nm MRX and the indicated concentration of 2-kb linear DNA. Kinase reactions were initiated with 5 nm Tel1 (lanes 1 and 2 are negative controls). Reactions were stopped after 15 min at 30 °C and analyzed by 7% SDS-PAGE, followed by phosphorimaging. Top panel, a representative experiment. Bottom panel, phosphorylation of Rad53 was quantified and plotted as -fold increase of Tel1 kinase activity with lane 3 set at 1. Averages and standard errors were derived from three independent experiments. B and C, time course analyses of Rad53 phosphorylation under standard conditions with 5 nm Tel1, 5 nm linear 2-kb DNA, and 30 nm MRX where indicated. D, quantification of the data in B. E, quantification of the data in C. Note the different y axis scales in D and E. A representative of two independent experiments is shown. In A–C, the migration of GST-Rad53kd (118 kDa), Xrs2 (96 kDa), and Mre11 (77 kDa) is as shown.
      To study the protein kinase activity of Tel1, we used the downstream effector kinase Rad53, which is a physiological Tel1 substrate (
      • Usui T.
      • Ogawa H.
      • Petrini J.H.
      A DNA damage response pathway controlled by Tel1 and the Mre11 complex.
      ). The catalytically inactive form Rad53kd was used so that all observable protein phosphorylation was Tel1-dependent. In addition, the general PIKK substrate PHAS-I was used in several experiments (
      • Brunn G.J.
      • Hudson C.C.
      • Sekulić A.
      • Williams J.M.
      • Hosoi H.
      • Houghton P.J.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.
      ), to query whether MRX stimulation of Tel1 kinase activity was specific for Rad53 as a substrate or a more general property.
      Fig. 1A summarizes the critical aspects of the assay, which show that Tel1 kinase activity is weakly stimulated by either DNA or MRX alone, but both DNA and MRX show strong synergism. This assay was followed by comprehensive time course analyses of each of the conditions. The weak activity of Tel1 alone (0.065 phosphates transferred to Rad53 per minute per Tel1 monomer) was stimulated about 2- to 3-fold in the presence of linear 2-kb plasmid DNA (Fig. 2, B and D). Without DNA, the MRX complex also increased Tel1’s basal activity 2- to 3-fold (Fig. 2, C and E). The presence of both DNA and MRX in the reaction resulted in a synergistic, 20- to 30-fold increase in Rad53 phosphorylation (Fig. 2, C and E). The synergistic stimulation of Tel1 by MRX and DNA is not specific to Rad53 as a substrate; it was also observed with PHAS-I, a commonly used substrate to probe the ATM/ATR family of kinases (Fig. S1A). In these assays, we had preassembled the MRX complex at 0 °C for 1 h prior to addition to the assay. To assess whether MRX preassembly is required, we either mixed the individually purified subunits of MRX at 0 °C for 1 h and added the pre-assembled complex to the assay or added them to the assay individually. In both cases, saturation was achieved at 10–20 nm MRX with 5 nm Tel1, indicating that preassembly was not required (Fig. S1, B and C). Therefore, we routinely used 30 nm MRX unless indicated otherwise.
      A titration of the kinase substrate PHAS-I in the Tel1 assay in the presence of MRX, either with or without DNA present, gave an approximately linear dose–response curve up to 20 μm PHAS-I, indicative of a weak association of this substrate with Tel1 (Fig. S1D). Given these results, we cannot conclude whether DNA enhances the affinity of kinase substrates for MRX·Tel1, which would be a possible explanation for the observed synergism. Finally, Tel1 also phosphorylates the Xrs2 and Mre11 subunits of MRX (Fig. 2A), as it does in the cell in response to DNA damage (
      • Nakada D.
      • Matsumoto K.
      • Sugimoto K.
      ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism.
      ,
      • Baroni E.
      • Viscardi V.
      • Cartagena-Lirola H.
      • Lucchini G.
      • Longhese M.P.
      The functions of budding yeast Sae2 in the DNA damage response require Mec1- and Tel1-dependent phosphorylation.
      ).

      Nucleosome-free dsDNA is the preferred effector for Tel1/MRX

      The homologous recombination repair pathway involves resection of double-stranded breaks by 5′-3′ exonucleases to generate ssDNA 3′ ends that are coated by the single-stranded DNA binding protein RPA (
      • Mimitou E.P.
      • Symington L.S.
      DNA end resection: many nucleases make light work.
      ). As resection proceeds and the 3′ overhangs increase in length, Tel1 activity is attenuated, and the checkpoint response is switched to that of a Mec1-mediated response (
      • Symington L.S.
      • Gautier J.
      Double-strand break end resection and repair pathway choice.
      ). Previous studies with human ATM and MRN showed that kilobase-long dsDNA substrates were required to detect stimulation of ATM kinase activity (
      • Lee J.H.
      • Paull T.T.
      ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
      ). Here the sensitivity of our in vitro kinase assay enabled us to evaluate the roles of DNA strandedness, structure, and length as effectors for Tel1. A single-stranded 80-mer oligonucleotide was inactive for stimulation, whether coated with RPA or not, whereas the double-stranded form showed significant activity (Fig. 3A). This is in direct contrast to Mec1, which shows optimal activity on RPA-coated ssDNA (
      • Majka J.
      • Niedziela-Majka A.
      • Burgers P.M.
      The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint.
      ). These findings are consistent with the proposed model where stretches of RPA-coated ssDNA resulting from DNA end resection mediate the transition from a Tel1- to a Mec1-dependent checkpoint response (
      • Shiotani B.
      • Zou L.
      Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks.
      ).
      Figure thumbnail gr3
      Figure 3MRX·Tel1 exhibits a preference for long dsDNA. A, standard kinase reactions contained 5 nm Tel1, 30 nm MRX, 200 nm Rad53, and the indicated concentrations of 80-mer ssDNA with or without RPA or 80-mer dsDNA. The amount of RPA required to coat ssDNA was calculated for each DNA concentration with a single RPA trimer coating 30 nt. Relative Tel1 activity was calculated by setting the reaction without DNA to 1 (lane 1). The first lane in the ssDNA/RPA experiment comes from a different gel in the same experiment. The gray bar indicates the discontinuity. The migration of GST-Rad53kd (118 kDa), Xrs2 (96 kDa), and Mre11 (77 kDa) is as shown. B, standard kinase reactions contained 5 nm Tel1, 30 nm MRX, 200 nm Rad53, and increasing concentrations of linear dsDNA molecules with the indicated lengths. Data were plotted as a function of DNA end concentrations in a log plot. Relative Tel1 kinase activity was calculated by setting the reaction without DNA to 1. Averages and standard errors were derived from four independent experiments. C, the same data were plotted as a function of total DNA as nucleotide concentrations.
      We next probed the DNA length requirement for stimulation. We compared double-stranded oligonucleotides in a size range of 20–80 bp, 147 bp, and a 2-kb linear plasmid DNA (Fig. 3, B and C). This analysis showed that, up to a length of 60 bp, there was little to no stimulation. The 147-mer dsDNA showed substantially higher activity than the 80-mer. A further increase in the effector DNA length to 2 kb showed another 2-fold increase in maximal activity. The activity data were plotted as a function of the concentration of dsDNA ends (Fig. 3B). Unexpectedly, the 2-kb linear DNA showed half-maximal activation at merely 0.1 nm DNA ends. If only DNA ends would be active in the MRX·Tel1 assay, given that the assay contains 2.5 nm Tel1 dimers, then half-maximal activation should occur at an end concentration of 1.25 nm DNA ends or more, depending on the affinity of MRX·Tel1 for ends. This titration experiment alone suggests that internal sites must be active for optimal stimulation of Tel1. The 147-mer dsDNA gave half-maximal activation at 0.2 nm ends and the 80-mer at 1.1 nm ends (Fig. 3B). Therefore, only the half-maximal concentration of the 80-mer is consistent with the model that only DNA ends serve as effectors of MRX·Tel1.
      The data were replotted as a function of the total nucleotide concentration (Fig. 3C). Half-maximal stimulation was reached at 45 nm, 16 nm, and 125 nm nucleotide concentration for the 80-mer, 147-mer, and 2-kb DNA, respectively. Although the total data indicate that some variation in the strength of DNA effectors may occur, possibly because of sequence and/or DNA composition contexts, they also indicate that internal sites serve as effectors of MRX·Tel1. Consequently, the large number of potential internal binding sites on the 2-kb DNA contribute substantially to stimulation.
      One possible model for Tel1 activity that maintains a requirement for DNA ends is one in which DNA ends are required for loading of MRX·Tel1 but that subsequently the complex can slide to internal regions along the dsDNA. This would allow assembly of additional complexes that are all active for kinase activity. This model would explain why the half-maximal concentration of the 2-kb effector is over 10 times lower than the concentration of MRX·Tel1 complexes. To test this model, we carried out an assay with the 2-kb plasmid either in the supercoiled, nicked, or linear form (Fig. 4A). In control experiments, we showed that the supercoiled and the nicked DNA effectors remained intact during the assay (data not shown). Mre11 is a known nuclease; however, Mre11 nuclease activity requires manganese, whereas our studies were carried out in magnesium-containing buffers (
      • Reginato G.
      • Cannavo E.
      • Cejka P.
      Physiological protein blocks direct the Mre11-Rad50-Xrs2 and Sae2 nuclease complex to initiate DNA end resection.
      ,
      • Trujillo K.M.
      • Yuan S.S.
      • Lee E.Y.
      • Sung P.
      Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95.
      ).
      Figure thumbnail gr4
      Figure 4Requirement for nucleosome-free DNA but not DNA ends for Tel1 activation. A, kinase reactions contained 5 nm Tel1, 30 nm MRX, 200 nm Rad53, and increasing concentrations of 2 kb DNA with the indicated structures. Relative Tel1 kinase activity was calculated by setting the reaction without DNA to 1. Averages and standard errors were derived from five independent experiments. The “no MRX” control titrations are shown for each DNA substrate. B, kinase reactions contained 5 nm Tel1, 30 nm MRX, 200 nm Rad53, and increasing concentrations of either linear or circular 262-bp DNA. Relative Tel1 kinase activity was calculated by setting the reaction without DNA to 1. Averages and standard errors were derived from three independent experiments. C, standard kinase reactions contained 5 nm Tel1, 30 nm MRX, 200 nm Rad53, and increasing concentrations of either 147-bp naked DNA or the nucleosome assembled on the same DNA. A representative experiment is shown. The migration of GST-Rad53kd (118 kDa), Xrs2 (96 kDa), and Mre11 (77 kDa) is as shown. D, quantification of four independent experiments with standard errors shown.
      Although the stimulation of Tel1 activity by MRX on linear DNA was slightly higher than on supercoiled or nicked DNA, the difference was of low statistical significance. Therefore, DNA ends are not essential for MRX-dependent Tel1 kinase activity. However, are ends stimulatory? The result shown in Fig. 4A would be expected if the contribution by the vast excess of internal sites masked the stimulatory contribution stemming from DNA ends. To reveal a possible contribution by DNA ends, we repeated this experiment with a much shorter DNA, 262 nt in length (Fig. S2A). Indeed, with this small DNA, the linear form is more active than the circular form, even though the circular DNA shows robust activity at higher DNA concentrations (Fig. 4B). Collectively, these data indicate the significance of internal DNA binding sites for MRX-dependent activation of Tel1. In support of the internal entry model, proficient internal loading of MRX has been shown in single-molecule studies (
      • Myler L.R.
      • Gallardo I.F.
      • Soniat M.M.
      • Deshpande R.A.
      • Gonzalez X.B.
      • Kim Y.
      • Paull T.T.
      • Finkelstein I.J.
      Single-molecule imaging reveals how Mre11-Rad50-Nbs1 initiates DNA break repair.
      ).
      So far, our experiments were carried out with naked DNA. We next determined whether nucleosomal DNA showed activity in our assay, but it did not. First, assembly of the DNA into a nucleosome caused inhibition of the MRX-independent but DNA-dependent activation of Tel1's basal kinase activity (Fig. 2A and Fig. S2B). With the complete system, we observed at least a 100-fold difference in the ability of the naked 147-mer to activate Tel1 compared with the same DNA wrapped up into a nucleosome (Fig. 4, C and D). The inhibitory effect of mononucleosomal DNA was recapitulated when PHAS-I was used instead of Rad53 as a substrate for Tel1 (Fig. S2C). We carried out two additional control experiments. We showed that histone octamers themselves, i.e. not bound up in a nucleosome, did not cause inhibition of Tel1 (Fig. S2D). Furthermore, we showed that the binding of DNA by general dsDNA binding proteins, such as HMGB-1, is also inhibitory, albeit less absolutely than that by the nucleosome (Fig. S2E).

      Rad50, but not Xrs2 or Mre11, is essential for DNA-dependent activation of Tel1 by MRX

      To determine which of the subunit(s) of the MRX complex are essential for Tel1 kinase activation, we measured Tel1 stimulation by each of the subunits alone and by the different heterodimeric complexes. These experiments were carried out in the presence of saturating concentrations of the 2-kb linear DNA effector. None of the single subunits alone stimulated Tel1 kinase (Fig. S3A), suggesting that the mere physical association of these factors with Tel1 (see below) is insufficient for activation. The Mre11–Xrs2 pair was also unable to stimulate kinase activity (Fig. 5A, lane 6). In contrast, both the Rad50–Mre11 and Rad50–Xrs2 pairs showed substantial kinase activation (Fig. 5A, lanes 8 and 10). These data indicate that although neither Mre11 or Xrs2 is required, the interaction of either subunit with Rad50 is a prerequisite for Rad50 to function in Tel1 activation. The activity of the complete MRX complex was 1.5-fold higher than that of the Rad50–Xrs2 pair. Two sets of observations exclude the possibility that the activity of the heterodimers is caused by cross-contamination of the Rad50–Mre11 assay with Xrs2 or the Rad50–Xrs2 subunit with Mre11. First, the Rad50–Mre11 assay lacks detectable phosphorylated Xrs2, and the Rad50–Xrs2 assay lacks detectable phosphorylated Mre11 (Fig. 5A, lanes 4 and 8, respectively). Second, the same robust activity of the Rad50–Xrs2 pair was observed when Rad50 was purified from a yeast mre11Δ mutant and Xrs2 from Escherichia coli, eliminating the possibility of contamination with Mre11 (Fig. S3B).
      Figure thumbnail gr5
      Figure 5Rad50 ATP binding is required for Tel1 activation. A, standard kinase reaction with 5 nm Tel1, 200 nm Rad53, and 30 nm MR, MX, RX, or MRX with or without 5 nm linear 2-kb DNA. Top panel, representative of three independent experiments. The right two lanes were derived from a different gel in the same experiment (both gels are shown in ). The gray bar indicates the discontinuity. Bottom panel, quantification and standard errors. Relative Tel1 kinase activity was calculated by setting Tel1 alone (lane 1) to 1. B, nitrocellulose filter binding assays of the binding of 50 μm [γ-32P]ATP to 500 nm WT or mutant MRX. Control, no MRX (see details under “Experimental procedures”). Quantification of four independent experiments with standard errors is shown. C, kinase assays were carried out using 5 nm Tel1; 200 nm Rad53; 30 nm MRX, MR(K40A)X, or MR(K40R)X; and increasing concentrations of 80-mer dsDNA. A representative of two experiments is shown. D, standard kinase reaction with 5 nm Tel1; 200 nm Rad53; 30 nm MRX, MR(K40A)X, or MR(K40R)X; 5 nm linear 2-kb DNA; and increasing concentrations of [γ-32P]ATP. Quantification of three independent experiments with standard errors is shown. In A and C, the migration of GST-Rad53kd (118 kDa), Xrs2 (96 kDa), and Mre11 (77 kDa) is as shown.
      Rad50 belongs to the ABC transporter family of ATPases and shares structural similarities to structural maintenance of chromosome proteins (
      • Paull T.T.
      20 Years of Mre11 biology: no end in sight.
      ). The ATP-dependent activities of S. cerevisiae Rad50 are required for all of its functions in DNA repair, as ATPase-dead mutants show a similar phenotype as rad50Δ mutants. DNA stimulates the ATPase activity of Rad50 alone or of the MRX complex, and at least one study shows that ends are important for this stimulation (
      • Deshpande R.A.
      • Lee J.H.
      • Paull T.T.
      Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites.
      ). We also observed that the linear plasmid was significantly more stimulatory for the ATPase than the circular plasmid DNA (Fig. S2F). As discussed below, ATP turnover is not important for MRX-dependent activation of Tel1 but ATP binding is. To test the ATP-binding requirement, we mutated the conserved Walker A-box lysine residue to either alanine or arginine, which has been shown to inactivate its ATPase activity (
      • Chen L.
      • Trujillo K.M.
      • Van Komen S.
      • Roh D.H.
      • Krejci L.
      • Lewis L.K.
      • Resnick M.A.
      • Sung P.
      • Tomkinson A.E.
      Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast.
      ). Neither mutant MRX complex showed detectable ATP binding (Fig. 5B). Furthermore, the mutant complexes were inactive for Tel1 stimulation (Fig. 5C). We further tested whether the Rad50–K40A and Rad50–K40R mutant MRX complexes retained partial activity in stimulating Tel1 at higher concentrations of ATP. However, even at ATP concentrations as high as 0.5 mm, no significant stimulation by the mutant complexes was observed (Fig. 5D). Thus, the ATP-dependent activity of Rad50 is essential for the stimulatory role of MRX.

      Each member of MRX interacts individually with Tel1

      To further increase the knowledge of the system, we probed the physical interactions between each of the MRX subunits with Tel1. The C-terminal region of Xrs2 has been shown to physically associate with Tel1, and this association is required for localizing Tel1 to DSBs (
      • Nakada D.
      • Matsumoto K.
      • Sugimoto K.
      ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism.
      ,
      • You Z.
      • Chahwan C.
      • Bailis J.
      • Hunter T.
      • Russell P.
      ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.
      ). To determine whether Mre11 and Rad50 also interact with Tel1, we performed a pulldown experiment with the individual subunits (Fig. 6A). GST-Tel1, or GST as a control, was incubated with each subunit together with GSH beads. The beads were washed three times with buffer to remove nonspecifically bound proteins and subjected to SDS-PAGE analysis. Proteins were visualized using silver staining (Fig. 6A). Tel1 was able to pull down each individual subunit, indicating that each subunit binds Tel1. Mre11 showed the most robust interaction with Tel1. As the Tel1-Rad50 interaction appeared rather weak, we repeated the pulldown experiment using a more sensitive Western blot analysis with anti-Rad50 antibodies (Fig. 6B, lane 5). This experiment positively established the interaction between Rad50 and Tel1. In addition, we probed Rad50 interactions with Mre11 and Xrs2 (Fig. 6B, lanes 3 and 4, respectively). As a positive control, we confirmed the known interaction of Rad50 with Mre11 (
      • Usui T.
      • Ohta T.
      • Oshiumi H.
      • Tomizawa J.
      • Ogawa H.
      • Ogawa T.
      Complex formation and functional versatility of Mre11 of budding yeast in recombination.
      ). A previous study established interactions between human Rad50 and Nbs1 (
      • van der Linden E.
      • Sanchez H.
      • Kinoshita E.
      • Kanaar R.
      • Wyman C.
      RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.
      ). Our data extend this interaction network to yeast Rad50 with Xrs2 (lane 4). These experiments establish that each of the subunits of MRX interact with each other and with Tel1.
      Figure thumbnail gr6
      Figure 6MRX·Tel1 interaction network. A, purified MRX subunits were incubated with GST-tagged Tel1 in the presence of glutathione-Sepharose beads (glut.-beads). The beads were washed, and bound proteins were analyzed by 7% SDS-PAGE, followed by silver staining (“Experimental procedures”). GST was used as a negative control for each subunit, as indicated. A representative of three independent experiments is shown. M, size markers. B, purified Rad50 subunit was incubated with either GST-tagged Mre11, Xrs2, or Tel1. Bound Rad50 was analyzed by Western blotting with anti-Rad50 antibody. Lane 2, negative control; binding of Rad50 to GST. C, model for MRX·Tel1 recruitment to non-nucleosomal DNA (see text for details).

      Discussion

      Prior to this biochemical analysis of the yeast MRX·Tel1 checkpoint complex, only the mammalian MRN·ATM complex had been studied in a purified protein system, in a series of insightful studies by Paull (
      • Paull T.T.
      Mechanisms of ATM Activation.
      ). Although a comparison of the yeast and mammalian systems shows some broad similarities, it also reveals critical differences that may have a decisive effect on the mechanism of action by these checkpoint complexes in the different organisms. Human ATM kinase activity is stimulated about 100-fold when both MRN and kilobase-long DNA are present, and there is an absolute requirement for dsDNA ends (
      • Lee J.H.
      • Paull T.T.
      ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
      ). There is also a requirement for Mn2+ rather than Mg2+; however, the nuclease activities of Mre11, which also depend on Mn2+, are dispensable for kinase activation (
      • Lee J.H.
      • Mand M.R.
      • Deshpande R.A.
      • Kinoshita E.
      • Yang S.H.
      • Wyman C.
      • Paull T.T.
      Ataxia telangiectasia-mutated (ATM) kinase activity is regulated by ATP-driven conformational changes in the Mre11/Rad50/Nbs1 (MRN) complex.
      ). ATM dimer-to-monomer transitions are mediated by phosphorylation and acetylation of specific residues (
      • Bakkenist C.J.
      • Kastan M.B.
      DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.
      ,
      • Sun Y.
      • Jiang X.
      • Chen S.
      • Fernandes N.
      • Price B.D.
      A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM.
      ). Based on mutational studies of ATM sites that are subject to posttranslational modification, it has been proposed that the active form of ATM is that of a monomer (
      • Lee J.H.
      • Paull T.T.
      ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
      ,
      • Bakkenist C.J.
      • Kastan M.B.
      DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.
      ). However, the orthologous mouse ATM mutants are without an ataxia telangiectasia phenotype, suggesting that regulation of ATM activity in human and mouse may not be conserved (
      • Pellegrini M.
      • Celeste A.
      • Difilippantonio S.
      • Guo R.
      • Wang W.
      • Feigenbaum L.
      • Nussenzweig A.
      Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo.
      ). Importantly, the assembly state of active ATM or Tel1 when bound to DNA still remains to be established.
      Our biochemical analysis was carried out under near-physiological conditions with Mg2+ as cofactor. The 50- to 100-fold stimulation of Tel1 kinase activity when both MRX and DNA are present allowed us to attach statistical significance to the observation that either MRX or DNA alone stimulates the kinase activity 2- to 3-fold (Fig. 2, D and E). Our study was aided by rigorous elimination of contaminating and cross-contaminating activities through isolation of the checkpoint proteins from appropriate yeast deletion strains (see “Results”). The dsDNA length requirement for activation was shorter for the yeast system, with nearly full activity achieved with a 147-mer DNA, whereas robust ATM activation required much longer DNA, 0.7 kb for partial activity and 2 kb for full activity (
      • Lee J.H.
      • Paull T.T.
      ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
      ). The fundamental difference between the human and the yeast system, however, is that MRX·Tel1 does not require DNA ends for maximal activity (Fig. 4A), whereas human MRN·ATM is absolutely dependent on DNA ends. The stimulation by DNA ends can only be observed on small DNA (Fig. 4B).
      In principle, this surprising result is not incompatible with the known dsDNA break checkpoint activity of MRX·Tel1. Kinase activation is completely inhibited on nucleosomal DNA (Fig. 4, C and D). This result predicts that spontaneous activation of Tel1 kinase would not occur on undamaged, fully chromatinized chromosomes. However, extensive nucleosome disassembly has been documented at dsDNA breaks in yeast and in other organisms (
      • Osley M.A.
      • Shen X.
      Altering nucleosomes during DNA double-strand break repair in yeast.
      ,
      • Tsukuda T.
      • Fleming A.B.
      • Nickoloff J.A.
      • Osley M.A.
      Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae.
      ). The resulting naked DNA is more than 0.5 kb in length in yeast, which is sufficient for robust MRX·Tel1 loading. This model of Tel1 activation at dsDNA breaks through its exclusion from normal chromatin is shown in Fig. 6C. The model does not exclude the possibility of other accessory factors further enhancing binding at dsDNA ends, but in our biochemical assay, there is no prerequisite for such factors.
      Structural and biochemical studies based on archaeal and bacterial Mre11–Rad50 complexes (
      • Stracker T.H.
      • Petrini J.H.
      The MRE11 complex: starting from the ends.
      ), as well as biochemical and genetic studies of eukaryotic MRX(N) complexes have led to a model where ATP binding by Rad50 promotes DNA binding and Tel1 kinase activation (
      • Deshpande R.A.
      • Lee J.H.
      • Paull T.T.
      Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites.
      ,
      • Cassani C.
      • Vertemara J.
      • Bassani M.
      • Marsella A.
      • Tisi R.
      • Zampella G.
      • Longhese M.P.
      The ATP-bound conformation of the Mre11-Rad50 complex is essential for Tel1/ATM activation.
      ). ATP hydrolysis, on the other hand, releases the complex from DNA (Fig. 1A). Consistent with this model, Rad50 ATP-binding mutants show no activation of Tel1 activity (Fig. 5C). As expected, our analysis of the individual subunits of MRX showed that Rad50 is essential for Tel1 activation in the presence of dsDNA (Fig. 5A). But it is not sufficient, and either Mre11 or Xrs2 is also required. Possibly, because Rad50 alone shows very weak interactions with Tel1 (Fig. 6A), the additional interactions mediated by either Mre11 or Xrs2 strengthen complex formation with Tel1, leading to robust kinase activation. In yeast, all three subunits of MRX are essential for Tel1-dependent phosphorylation of Rad53 in response to zeocin, a dsDNA break–inducing agent, although Xrs2 is dispensable for several other functions of MRX, provided nuclear localization of Mre11–Rad50 is ensured (
      • Oh J.
      • Al-Zain A.
      • Cannavo E.
      • Cejka P.
      • Symington L.S.
      Xrs2 dependent and independent functions of the Mre11-Rad50 complex.
      ).
      When comparing the activation mechanism of Tel1ATM with that of the related PIKKs Mec1ATR and DNA-PK, it is interesting to note that Mec1 can be activated by an activator protein alone, such as Dpb11TopBP1, or even by a peptide derived from an activator protein (
      • Wanrooij P.H.
      • Tannous E.
      • Kumar S.
      • Navadgi-Patil V.M.
      • Burgers P.M.
      Probing the Mec1ATR checkpoint activation mechanism with small peptides.
      ), whereas DNA-PK activation can be accomplished by DNA binding alone (
      • Poltoratsky V.P.
      • Shi X.
      • York J.D.
      • Lieber M.R.
      • Carter T.H.
      Human DNA-activated protein kinase (DNA-PK) is homologous to phosphatidylinositol kinases.
      ). In contrast, Tel1 requires both DNA and MRX, with Rad50 also requiring proficient ATP-binding capacity. A comprehensive description of the Tel1 activation mechanism will have to take into consideration that DNA and MRX individually stimulate Tel1 kinase activity 2- to 3-fold, but that both factors together show a profound synergism. Our studies show that Tel1 alone binds DNA proficiently and saturably in the absence of MRX (Fig. 2D), and Tel1 binds MRX proficiently in the absence of DNA (Fig. 6, A and B). Therefore, increases in binding affinities are unlikely explanations for the observed hyperactivity by the ternary complex. It is probable that DNA-induced conformational changes within the MRX complex are the driving force for Tel1 kinase activation. Whether kinase activation is a consequence of MRX- and DNA-mediated monomerization of Tel1 or whether other MRX- and DNA-induced conformational rearrangements of Tel1 result in its activation is currently not known and an important question for further investigation.

      Experimental procedures

      Yeast strains, DNA, and proteins

      The strains used were BJ2168 (MATa leu2–3,112 pep4–3 prb1–1122 prc1–407 trp1–289 ura3–52), PY265 (MATa can1 his3 leu2 trp1 ura3 pep4Δ::HIS3 nam7Δ::KanMX4), PY335 (MATa his3 leu2 trp1 ura3 pep4Δ::HIS3 tel1Δ::KanMX4 tda1Δ::NAT1), and BY4741-Mre11 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 mre11Δ::KanMX4). The oligonucleotides used are listed in the supporting text.
      Tel1 was overexpressed in yeast strain PY265 from the galactose-inducible plasmid pBL602 and purified as described previously (
      • Sawicka M.
      • Wanrooij P.H.
      • Darbari V.C.
      • Tannous E.
      • Hailemariam S.
      • Bose D.
      • Makarova A.V.
      • Burgers P.M.
      • Zhang X.
      The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organization.
      ). The protein was purified containing an N-terminal GST domain fusion. The GST domain was retained for the protein interaction studies in Fig. 6A but was cleaved off followed by heparin-agarose purification, as described previously (
      • Sawicka M.
      • Wanrooij P.H.
      • Darbari V.C.
      • Tannous E.
      • Hailemariam S.
      • Bose D.
      • Makarova A.V.
      • Burgers P.M.
      • Zhang X.
      The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organization.
      ), for all other experiments. Rad50 was overexpressed in BJ2168 or BY4741–Mre11 from the galactose-inducible plasmid pPM321 and purified as described previously (
      • Trujillo K.M.
      • Sung P.
      DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex.
      ). Rad50 mutants were made in this plasmid and purified similarly.
      Plasmid pBL535 (pRS424-GAL (2 μm origin, TRP1) ZZ-3C-XRS2, MRE11) has the IgG-binding StrepA-ZZ tag fused to the N terminus of Xrs2, separated by a Prescission protease cleavage site, and Mre11 divergently expressed from the galactose-inducible GAL1–10 promoter. The IgG-tagged Mre11–Xrs2 dimer was overexpressed in yeast strain PY335 from pBL535. After growth and galactose induction, cells were harvested and resuspended in a half-volume of 3× Hep200 buffer (30 mm HEPES (pH 7.8), 10% glycerol, 200 mm NaCl, 150 mm ammonium sulfate, 2 mm DTT, 0.1% Tween 20, 1 mm EDTA, 0.5 mm EGTA, 5 μm pepstatin A, 5 μm leupeptin, and 1 mm NaHSO3; the suffix refers to mm NaCl in the buffer) and frozen in liquid nitrogen as popcorn as described previously (
      • Bylund G.O.
      • Majka J.
      • Burgers P.M.
      Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae.
      ). The frozen cells were lysed by blending in dry ice in, treated with polyamin P, and centrifuged, and the supernatant was treated with ammonium sulfate and centrifuged as described previously (
      • Bylund G.O.
      • Majka J.
      • Burgers P.M.
      Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae.
      ).The ammonium sulfate pellet was resuspended in HEP0 buffer (as HEP200 buffer but without NaCl) and incubated with IgG-Sepharose beads (GE Healthcare, 1 ml/100 g of frozen popcorn cells) for 4 h at 4 °C. The beads were washed with 50 column volumes of HEP300 supplemented with 0.02% E10C12 detergent. The beads were rotated gently overnight at 4 °C with 1 ml of HEP300 supplemented with Prescission protease to cleave the ZZ tag. The Mre11–Xrs2 dimer was further purified over a 1-ml heparin-agarose column. The protein was loaded in HEP300 buffer, the column was washed with 50 ml HEP300, and the protein was eluted with HEP700 buffer.
      Plasmid pBL532 (pRS424-GAL (2 μm ori, TRP1) GST-3C-MRE11) has GST fusion at the N terminus of Mre11, separated by a Prescission protease cleavage site, placed under control of the galactose-inducible GAL1–10 promoter. Expression of Mre11 in yeast strain PY335 and purification were exactly as described for Mre11–Xrs2. Xrs2 was overexpressed in E. coli BL21-DE3-Star (Invitrogen) from plasmid pBL533 (pGEX-6P1-GST-3C-Xrs2). Cells were grown and induced at 37 °C using standard procedures. The cells were harvested and resuspended in an equal volume of 2× HEP200 buffer (including 5 μm bestatin and 1 μm E64, both from Sigma-Aldrich), subjected to a freeze–thaw cycle, incubated with 0.2 mg/ml lysozyme for 30 min on ice, and lysed by sonication. Polyamin P (40 μl of a 10% solution per milliliter of lysate) was added, and the lysate was cleared at 27,000 × g for 20 min. Solid ammonium sulfate (0.3 g per milliliter of cleared lysate) was added with stirring, and after 30 min, the precipitate was collected at 27,000 × g for 30 min and resuspended in 10 ml (per liter of induction culture) of the same buffer. After binding for 4 h to 1 ml of GSH-Sepharose, the beads were washed with 50 ml of buffer, followed by elution with 30 mm reduced GSH in HEP200 (but at pH 8.1). The eluted Xrs2 was further purified over a 1-ml heparin agarose column as described above. The MRX complex was formed by incubating equimolar amounts of Rad50 and Mre11–Xrs2 or the individual subunits at 0 °C for 1 h.

      Tel1 kinase assay

      The 10-μl standard assay contained 25 mm HEPES-NaOH (pH 7.6), 80 mm NaCl, 7 mm Mg acetate, 100 μg/ml BSA, 1 mm DTT, 50 μm ATP, 0.05 μCi [γ-32P]ATP, 200 nm GST-Rad53kd, 30 nm MRX, and 5 nm linear 2-kb DNA (pUC19-Δ2, a 1973-bp plasmid derived from pUC19 cut with BamHI). Reactions were started with 5 nm Tel1 (monomer concentration) at 30 °C for 15 min, stopped with 4 ml of 2.5× SDS-PAGE loading dye, boiled, and separated on 7% SDS-PAGE gels. The gels were dried and exposed to a phosphor screen (GE Healthcare). Variations are indicated in the figure legends. pUC19-Δ2 was nicked with the BbvC1 nickase (New England Biolabs) for the experiment shown in Fig. 4A. Quantification of the data was carried out using ImageQuant software. Data were analyzed and plotted using KaleidaGraph software, which was also used to model the data in Figs. 3, B and C, and 5D to the Michaelis–Menten equation

      GST pulldown experiments and ATP-binding studies

      Purified MRX subunits (500 ng) were incubated with GST-tagged Tel1 (2 μg) in the presence of 50 μl of GSH-Sepharose beads in a total volume of 200 μl of HEP200 buffer (without protease inhibitors). After 4 h of incubation at 4 °C with rotation, the beads were subjected to three 0.5-ml washes with HEP200 to remove nonspecifically bound proteins. The beads were loaded in a spin column, centrifuged briefly to remove residual buffer, suspended in 50 μl of SDS-PAGE loading buffer, boiled, and spun, and the supernatant was analyzed by 7% SDS-PAGE. Visualization was by silver staining. Alternatively, the proteins were transferred to a PVDF membrane and processed further for Western blot analysis with anti-scRad50 antibodies (Thermo Fisher) using standard procedures.
      Nitrocellulose filter binding experiments were performed as described previously (
      • Gomes X.V.
      • Schmidt S.L.
      • Burgers P.M.
      ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.
      ) using 500 nm MRX in kinase buffer and 50 μm [γ-32P]ATP in a 20-μl assay. After incubation at 0 °C for 30 min, the mixture was passed through a nitrocellulose membrane with gentle suction and washed with 2 × 0.5 ml of buffer. The dried filters were subjected to Cerenkov scintillation counting.

      Author contributions

      S. H., S. K., and P. M. B. conceptualization; S. H., S. K., and P. M. B. data curation; S. H. and P. M. B. formal analysis; S. H. and P. M. B. investigation; S. H. and P. M. B. writing - original draft; S. H., S. K., and P. M. B. writing - review and editing; P. M. B. resources; P. M. B. supervision; P. M. B. funding acquisition; P. M. B. project administration.

      Acknowledgments

      We thank Bonita Yoder for strain construction, Carrie Stith for general support with all aspects of this work, and Roberto Galletto for the Xrs2 overexpression plasmid.

      Supplementary Material

      References

        • Lavin M.F.
        • Shiloh Y.
        The genetic defect in ataxia-telangiectasia.
        Annu. Rev. Immunol. 1997; 15 (9143686): 177-202
        • Oh J.
        • Symington L.S.
        Role of the Mre11 complex in preserving genome integrity.
        Genes. 2018; 9 (30501098): E589
        • Abraham R.T.
        Cell cycle checkpoint signaling through the ATM and ATR kinases.
        Genes Dev. 2001; 15 (11544175): 2177-2196
        • Mallory J.C.
        • Petes T.D.
        Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (11095737): 13749-13754
        • Perry J.
        • Kleckner N.
        The ATRs, ATMs, and TORs are giant HEAT repeat proteins.
        Cell. 2003; 112 (12553904): 151-155
        • Sawicka M.
        • Wanrooij P.H.
        • Darbari V.C.
        • Tannous E.
        • Hailemariam S.
        • Bose D.
        • Makarova A.V.
        • Burgers P.M.
        • Zhang X.
        The dimeric architecture of checkpoint kinases Mec1ATR and Tel1ATM reveal a common structural organization.
        J. Biol. Chem. 2016; 291 (27129217): 13436-13447
        • Shiloh Y.
        ATM and related protein kinases: safeguarding genome integrity.
        Nat. Rev. Cancer. 2003; 3 (12612651): 155-168
        • Bosotti R.
        • Isacchi A.
        • Sonnhammer E.L.
        FAT: a novel domain in PIK-related kinases.
        Trends Biochem. Sci. 2000; 25 (10782091): 225-227
        • Ogi H.
        • Goto G.H.
        • Ghosh A.
        • Zencir S.
        • Henry E.
        • Sugimoto K.
        Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition.
        Mol. Biol. Cell. 2015; 26 (26246601): 3480-3488
        • Wang X.
        • Chu H.
        • Lv M.
        • Zhang Z.
        • Qiu S.
        • Liu H.
        • Shen X.
        • Wang W.
        • Cai G.
        Structure of the intact ATM/Tel1 kinase.
        Nat. Commun. 2016; 7 (27229179)11655
        • Sanchez Y.
        • Desany B.A.
        • Jones W.J.
        • Liu Q.
        • Wang B.
        • Elledge S.J.
        Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways.
        Science. 1996; 271 (8553072): 357-360
        • Usui T.
        • Ogawa H.
        • Petrini J.H.
        A DNA damage response pathway controlled by Tel1 and the Mre11 complex.
        Mol. Cell. 2001; 7 (11430828): 1255-1266
        • Zou L.
        • Elledge S.J.
        Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
        Science. 2003; 300 (12791985): 1542-1548
        • Downs J.A.
        • Lowndes N.F.
        • Jackson S.P.
        A role for Saccharomyces cerevisiae histone H2A in DNA repair.
        Nature. 2000; 408 (11140636): 1001-1004
        • Hammet A.
        • Magill C.
        • Heierhorst J.
        • Jackson S.P.
        Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.
        EMBO Rep. 2007; 8 (17721446): 851-857
        • Usui T.
        • Foster S.S.
        • Petrini J.H.
        Maintenance of the DNA damage checkpoint requires DNA damage-induced mediator protein oligomerization.
        Mol. Cell. 2009; 33 (19187758): 147-159
        • Lee S.J.
        • Schwartz M.F.
        • Duong J.K.
        • Stern D.F.
        Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling.
        Mol. Cell. Biol. 2003; 23 (12917350): 6300-6314
        • Nakada D.
        • Matsumoto K.
        • Sugimoto K.
        ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism.
        Genes Dev. 2003; 17 (12923051): 1957-1962
        • You Z.
        • Chahwan C.
        • Bailis J.
        • Hunter T.
        • Russell P.
        ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.
        Mol. Cell. Biol. 2005; 25 (15964794): 5363-5379
        • Aravind L.
        • Walker D.R.
        • Koonin E.V.
        Conserved domains in DNA repair proteins and evolution of repair systems.
        Nucleic Acids Res. 1999; 27 (9973609): 1223-1242
        • Adelman C.A.
        • Petrini J.H.
        Division of labor: DNA repair and the cell cycle specific functions of the Mre11 complex.
        Cell Cycle. 2009; 8 (19395852): 1510-1514
        • Lamarche B.J.
        • Orazio N.I.
        • Weitzman M.D.
        The MRN complex in double-strand break repair and telomere maintenance.
        FEBS Lett. 2010; 584 (20655309): 3682-3695
        • Paull T.T.
        • Gellert M.
        The 3′ to 5′ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks.
        Mol. Cell. 1998; 1 (9651580): 969-979
        • Usui T.
        • Ohta T.
        • Oshiumi H.
        • Tomizawa J.
        • Ogawa H.
        • Ogawa T.
        Complex formation and functional versatility of Mre11 of budding yeast in recombination.
        Cell. 1998; 95 (9845372): 705-716
        • Furuse M.
        • Nagase Y.
        • Tsubouchi H.
        • Murakami-Murofushi K.
        • Shibata T.
        • Ohta K.
        Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.
        EMBO J. 1998; 17 (9799249): 6412-6425
        • Symington L.S.
        • Gautier J.
        Double-strand break end resection and repair pathway choice.
        Annu. Rev. Genet. 2011; 45 (21910633): 247-271
        • Hopfner K.P.
        • Karcher A.
        • Shin D.S.
        • Craig L.
        • Arthur L.M.
        • Carney J.P.
        • Tainer J.A.
        Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.
        Cell. 2000; 101 (10892749): 789-800
        • Raymond W.E.
        • Kleckner N.
        RAD50 protein of S. cerevisiae exhibits ATP-dependent DNA binding.
        Nucleic Acids Res. 1993; 21 (8367302): 3851-3856
        • Melby T.E.
        • Ciampaglio C.N.
        • Briscoe G.
        • Erickson H.P.
        The symmetrical structure of structural maintenance of chromosomes (SMC) and MukB proteins: long, antiparallel coiled coils, folded at a flexible hinge.
        J. Cell Biol. 1998; 142 (9744887): 1595-1604
        • Walker J.E.
        • Saraste M.
        • Runswick M.J.
        • Gay N.J.
        Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.
        EMBO J. 1982; 1 (6329717): 945-951
        • Hopfner K.P.
        • Craig L.
        • Moncalian G.
        • Zinkel R.A.
        • Usui T.
        • Owen B.A.
        • Karcher A.
        • Henderson B.
        • Bodmer J.L.
        • McMurray C.T.
        • Carney J.P.
        • Petrini J.H.
        • Tainer J.A.
        The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair.
        Nature. 2002; 418 (12152085): 562-566
        • Lammens K.
        • Bemeleit D.J.
        • Möckel C.
        • Clausing E.
        • Schele A.
        • Hartung S.
        • Schiller C.B.
        • Lucas M.
        • Angermüller C.
        • Söding J.
        • Strässer K.
        • Hopfner K.P.
        The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair.
        Cell. 2011; 145 (21458667): 54-66
        • Möckel C.
        • Lammens K.
        • Schele A.
        • Hopfner K.P.
        ATP driven structural changes of the bacterial Mre11:Rad50 catalytic head complex.
        Nucleic Acids Res. 2012; 40 (21937514): 914-927
        • Anderson D.E.
        • Trujillo K.M.
        • Sung P.
        • Erickson H.P.
        Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy.
        J. Biol. Chem. 2001; 276 (11470800): 37027-37033
        • de Jager M.
        • van Noort J.
        • van Gent D.C.
        • Dekker C.
        • Kanaar R.
        • Wyman C.
        Human Rad50/Mre11 is a flexible complex that can tether DNA ends.
        Mol. Cell. 2001; 8 (11741547): 1129-1135
        • Hopfner 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 (11371344): 473-485
        • Moreno-Herrero F.
        • de Jager M.
        • Dekker N.H.
        • Kanaar R.
        • Wyman C.
        • Dekker C.
        Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA.
        Nature. 2005; 437 (16163361): 440-443
        • Paull T.T.
        20 Years of Mre11 biology: no end in sight.
        Mol. Cell. 2018; 71 (30057197): 419-427
        • Lau W.C.
        • Li Y.
        • Liu Z.
        • Gao Y.
        • Zhang Q.
        • Huen M.S.
        Structure of the human dimeric ATM kinase.
        Cell Cycle. 2016; 15 (27097373): 1117-1124
        • Reid R.J.
        • González-Barrera S.
        • Sunjevaric I.
        • Alvaro D.
        • Ciccone S.
        • Wagner M.
        • Rothstein R.
        Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I-induced DNA damage.
        Genome Res. 2011; 21 (21173034): 477-486
        • Brunn G.J.
        • Hudson C.C.
        • Sekulić A.
        • Williams J.M.
        • Hosoi H.
        • Houghton P.J.
        • Lawrence Jr., J.C.
        • Abraham R.T.
        Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.
        Science. 1997; 277 (9204908): 99-101
        • Baroni E.
        • Viscardi V.
        • Cartagena-Lirola H.
        • Lucchini G.
        • Longhese M.P.
        The functions of budding yeast Sae2 in the DNA damage response require Mec1- and Tel1-dependent phosphorylation.
        Mol. Cell. Biol. 2004; 24 (15121837): 4151-4165
        • Mimitou E.P.
        • Symington L.S.
        DNA end resection: many nucleases make light work.
        DNA Repair. 2009; 8 (19473888): 983-995
        • Lee J.H.
        • Paull T.T.
        ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.
        Science. 2005; 308 (15790808): 551-554
        • Majka J.
        • Niedziela-Majka A.
        • Burgers P.M.
        The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint.
        Mol. Cell. 2006; 24 (17189191): 891-901
        • Shiotani B.
        • Zou L.
        Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks.
        Mol. Cell. 2009; 33 (19285939): 547-558
        • Reginato G.
        • Cannavo E.
        • Cejka P.
        Physiological protein blocks direct the Mre11-Rad50-Xrs2 and Sae2 nuclease complex to initiate DNA end resection.
        Genes Dev. 2017; 31 (29321179): 2325-2330
        • Trujillo K.M.
        • Yuan S.S.
        • Lee E.Y.
        • Sung P.
        Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95.
        J. Biol. Chem. 1998; 273 (9705271): 21447-21450
        • Myler L.R.
        • Gallardo I.F.
        • Soniat M.M.
        • Deshpande R.A.
        • Gonzalez X.B.
        • Kim Y.
        • Paull T.T.
        • Finkelstein I.J.
        Single-molecule imaging reveals how Mre11-Rad50-Nbs1 initiates DNA break repair.
        Mol. Cell. 2017; 67 (28867292): 891-898.e4
        • Deshpande R.A.
        • Lee J.H.
        • Paull T.T.
        Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites.
        Nucleic Acids Res. 2017; 45 (28369545): 5255-5268
        • Chen L.
        • Trujillo K.M.
        • Van Komen S.
        • Roh D.H.
        • Krejci L.
        • Lewis L.K.
        • Resnick M.A.
        • Sung P.
        • Tomkinson A.E.
        Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast.
        J. Biol. Chem. 2005; 280 (15546877): 2620-2627
        • van der Linden E.
        • Sanchez H.
        • Kinoshita E.
        • Kanaar R.
        • Wyman C.
        RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.
        Nucleic Acids Res. 2009; 37 (19151086): 1580-1588
        • Paull T.T.
        Mechanisms of ATM Activation.
        Annu. Rev. Biochem. 2015; 84 (25580527): 711-738
        • Lee J.H.
        • Mand M.R.
        • Deshpande R.A.
        • Kinoshita E.
        • Yang S.H.
        • Wyman C.
        • Paull T.T.
        Ataxia telangiectasia-mutated (ATM) kinase activity is regulated by ATP-driven conformational changes in the Mre11/Rad50/Nbs1 (MRN) complex.
        J. Biol. Chem. 2013; 288 (23525106): 12840-12851
        • Bakkenist C.J.
        • Kastan M.B.
        DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.
        Nature. 2003; 421 (12556884): 499-506
        • Sun Y.
        • Jiang X.
        • Chen S.
        • Fernandes N.
        • Price B.D.
        A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (16141325): 13182-13187
        • Pellegrini M.
        • Celeste A.
        • Difilippantonio S.
        • Guo R.
        • Wang W.
        • Feigenbaum L.
        • Nussenzweig A.
        Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo.
        Nature. 2006; 443 (16906133): 222-225
        • Osley M.A.
        • Shen X.
        Altering nucleosomes during DNA double-strand break repair in yeast.
        Trends Genet. 2006; 22 (16997415): 671-677
        • Tsukuda T.
        • Fleming A.B.
        • Nickoloff J.A.
        • Osley M.A.
        Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae.
        Nature. 2005; 438 (16292314): 379-383
        • Stracker T.H.
        • Petrini J.H.
        The MRE11 complex: starting from the ends.
        Nat. Rev. Mol. Cell Biol. 2011; 12 (21252998): 90-103
        • Cassani C.
        • Vertemara J.
        • Bassani M.
        • Marsella A.
        • Tisi R.
        • Zampella G.
        • Longhese M.P.
        The ATP-bound conformation of the Mre11-Rad50 complex is essential for Tel1/ATM activation.
        Nucleic Acids Res. 2019; 47 (30698745): 3550-3567
        • Oh J.
        • Al-Zain A.
        • Cannavo E.
        • Cejka P.
        • Symington L.S.
        Xrs2 dependent and independent functions of the Mre11-Rad50 complex.
        Mol. Cell. 2016; 64 (27746018): 405-415
        • Wanrooij P.H.
        • Tannous E.
        • Kumar S.
        • Navadgi-Patil V.M.
        • Burgers P.M.
        Probing the Mec1ATR checkpoint activation mechanism with small peptides.
        J. Biol. Chem. 2016; 291 (26499799): 393-401
        • Poltoratsky V.P.
        • Shi X.
        • York J.D.
        • Lieber M.R.
        • Carter T.H.
        Human DNA-activated protein kinase (DNA-PK) is homologous to phosphatidylinositol kinases.
        J. Immunol. 1995; 155 (7594449): 4529-4533
        • Trujillo K.M.
        • Sung P.
        DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex.
        J. Biol. Chem. 2001; 276 (11454871): 35458-35464
        • Bylund G.O.
        • Majka J.
        • Burgers P.M.
        Overproduction and purification of RFC-related clamp loaders and PCNA-related clamps from Saccharomyces cerevisiae.
        Methods Enzymol. 2006; 409 (16793392): 1-11
        • Gomes X.V.
        • Schmidt S.L.
        • Burgers P.M.
        ATP utilization by yeast replication factor C. II. Multiple stepwise ATP binding events are required to load proliferating cell nuclear antigen onto primed DNA.
        J. Biol. Chem. 2001; 276 (11432856): 34776-34783