Regulation of protein kinase Tel1 after phleomycin treatment. (A) Tel1 activation after phleomycin treatment. Wild-type (WT) and tel1-KN mutant cells expressing HA-tagged Tel1 protein were incubated with nocodazole to arrest at G₂/M. Cells were then either mock treated (−) or treated (+) with 20 μg/ml phleomycin (PHL) for 60 min. Immunoprecipitated HA-Tel1 was subjected to the in vitro kinase assay using GST-Rad53, a recombinant protein purified from E. coli, as a substrate. Incorporation of ³²P into GST-Rad53 was monitored by phosphorimaging. The amounts of HA-Tel1 and Mre11 in immunoprecipitates were examined by immunoblotting with an anti-HA or anti-Mre11 antibody. The amount of DNA at the SMC2 locus in immunoprecipitates was analyzed by real-time PCR and, for each immunoprecipitate, was normalized to the amount from mock-treated wild-type cells. Phosphorylation of GST-Rad53 was normalized to that observed with immunoprecipitated wild-type Tel1 kinase after mock treatment. Relative phosphorylation was determined from three independent experiments. Bars represent averages; error bars, standard deviations. (B) Tel1 activation kinase after incubation with various concentrations of phleomycin. Cells were treated with various concentrations of phleomycin and were subjected to the in vitro kinase assay as for panel A. (C) Effect of xrs2-11 mutation on Tel1 activation after phleomycin treatment. Wild-type and xrs2-11 mutant cells expressing HA-tagged Tel1 protein were subjected to the in vitro kinase assay as for panel A. (D) Effect of tel1-KN mutation on the association of Tel1 with DSBs. Cells expressing HA-Tel1 or HA-Tel1-KN were transformed with the GAL-HO plasmid. (Top) The strains contain an HO cleavage site, marked with HIS2, at the ADH4 locus on chromosome (Chr.) VII. The HO1 primer pair amplifies a region 1 kb away from the HO cleavage site. An arrow represents the telomere. (Bottom) Transformed cells were analyzed for association of Tel1 with HO-induced DSBs by real-time PCR. Relative enrichment was determined from three independent experiments. Bars represent averages; error bars, standard deviations.

Effect of sae2Δ, mre11-3, or rad1Δ tdp1Δ mutation on activation of protein kinase Tel1 after CPT treatment. (A) Effect of mre11 or sae2 mutation on sensitivity to CPT. Serial dilutions of cultures of wild-type (WT), mre11Δ, mre11-3, or sae2Δ mutant cells were spotted onto medium containing various concentrations of CPT. (B) Effect of mre11-3 or sae2Δ mutation on accumulation of Top1-DNA complexes on chromosomes after CPT treatment. Wild-type, mre11-3, and sae2Δ mutant cells expressing FLAG-tagged Top1 were arrested at G₁ with α-factor. After arrest, cells were washed and released into the medium in the presence or absence of 100 μM CPT. Cells were collected 30 min after release. Extracts were fractionated on a CsCl gradient. (Top) Fractions containing chromosomal DNA were subjected to immunoblot analysis with anti-FLAG antibodies. The amount of DNA at the SMC2 locus was analyzed by real-time PCR and, for each fraction, was normalized to that from mock-treated wild-type cells. (Bottom) The relative increase in the level of covalently attached Top1 was determined by normalizing Top1 accumulation in the DNA fraction from a particular strain to that for wild-type cells after CPT treatment. Top1 accumulation is defined as the amount of Top1 immunoblot signals relative to the amount of PCR amplification signals for the SMC2 locus. Two independent experiments were performed. (C) Effect of sae2Δ or mre11-3 mutation on activation of protein kinase Tel1 after CPT treatment. Wild-type, sae2Δ, and mre11-3 cells expressing HA-tagged Tel1 were cultured as for panel B and were treated with 20 μM CPT. Cells were then analyzed as for Fig. 1A. (D) Effect of mre11-3 or sae2Δ mutation on Tel1-mediated phosphorylation of Rad53 after CPT treatment. Wild-type, mec1Δ, mec1Δ mre11-3, and mec1Δ sae2Δ mutant cells expressing HA-tagged Rad53 protein were treated with 20 μM CPT as for panel C and were subjected to immunoblot analysis with anti-HA antibodies. (E) Effect of rad1Δ tdp1Δ mutation on accumulation of stable Top1-DNA complexes after CPT treatment. Wild-type and rad1Δ tdp1Δ mutant cells expressing FLAG-tagged Top1 were analyzed as for panel B. (F) Effect of sae2Δ mutation or rad1Δ tdp1Δ mutation on activation of protein kinase Tel1 after treatment with various concentrations of CPT. Wild-type, sae2Δ, or rad1Δ tdp1Δ cells expressing HA-tagged Tel1 were treated with various concentrations of CPT as for panel B and were analyzed as for Fig. 2A.

Effect of a Fab-blocked DNA end on Tel1 catalytic activity in vitro. (A) Schematic of the DNA fragments used for the in vitro reconstitution. The DNA fragment designated 5′ or 3′ contains biotinylated dCTP at the 5′ or 3′ end, respectively. DNA fragment I is internally labeled with biotinylated dCTP. The control DNA fragment N or N1 has no biotinylation. Biotinylated DNA fragments are bound by anti-biotin Fab fragments (filled circles). The sequence of DNA fragment N is the same as that of DNA fragment 5′ or 3′, whereas the sequence of DNA fragment N1 is the same as that of DNA fragment I. All the DNA fragments are 150 bp long. (B and C) Effect of Fab tethering on MRX-mediated DNA degradation. A 150-bp DNA fragment (N, 5′, 3′, N1, or I) was incubated with MRX in the presence of Mn²⁺ for 10 min (B) or as long as 30 min (C). DNA species were separated by electrophoresis and were detected by phosphorimaging analysis. (D) Effects of Fab-blocked DNA ends on DNA binding of the MRX complex. A DNA fragment (N, 5′, 3′, N1, or I) was incubated with MRX in the absence of Mn²⁺ and was precipitated by using an anti-Xrs2 antibody. The amount of precipitated DNA was analyzed by real-time PCR. Relative enrichment was determined from three independent experiments. Bars represent averages; error bars, standard deviations. (E) Effects of blocked DNA ends on Tel1 activation. Tel1 or Tel1-KN protein was incubated with the MRX complex or one of the various DNA fragments, and Tel1 catalytic activity was determined as for Fig. 4A. Fab-bound DNA fragments were prepared by mixing Fab and DNA fragments prior to the kinase reaction.

Effect of DNA end processing on protein kinase activity of Tel1 in vitro. (A) MRX-dependent DNA degradation. Tel1 protein, the MRX complex (either wild-type Mre11 or the Mre11-3 mutant), and a ³²P-labeled DNA fragment (N or 5′) were incubated in the reaction buffer as indicated. After incubation, the reaction mixtures were treated with proteinase K, and DNA species were analyzed as for Fig. 5B. (B) Effect of DNA end processing on Tel1 catalytic activity in the two-step in vitro reaction. Tel1 protein, the MRX complex, and an unlabeled DNA fragment were first incubated in the reaction buffer for different times, and [³²P]ATP and GST-Rad53 were added later. After incubation with GST-Rad53 for 10 min, the reaction was stopped at the indicated time, and the results were analyzed as for Fig. 4A. The phosphorylation levels of GST-Rad53 were normalized to that observed in the reaction carried out in the absence of MRX or DNA for 10 min.

Effect of sae2Δ mutation on Tel1 activation and histone-DNA complex accumulation. (A) Effect of sae2Δ mutation on activation of protein kinase Tel1 after phleomycin (PHL) treatment. Wild-type (WT) and sae2Δ mutant cells expressing HA-tagged Tel1 were analyzed as for Fig. 1A. (B) Effect of sae2Δ mutation on Tel1-mediated phosphorylation of Rad9 and Rad53 after phleomycin treatment. Wild-type, mec1Δ, and mec1Δ sae2Δ mutant cells expressing HA-tagged Rad9 or Rad53 protein were treated with phleomycin as for panel A and were subjected to immunoblot analysis with anti-HA antibodies. (C) Effect of sae2Δ mutation on Tel1 activation after EcoRI expression. Wild-type and sae2Δ mutant cells expressing HA-tagged Tel1 were transformed with pGAL-EcoRI (+) or the control vector (−). Transformed cells were grown in sucrose to repress EcoRI expression and were then synchronized at G₂/M phase with nocodazole. After arrest, the culture was incubated with galactose to induce EcoRI expression for 120 min. EcoRI-expressing sae2Δ cells were additionally treated with 20 μg/ml phleomycin in galactose for 60 min. Cells were analyzed as for panel A. (D) Degradation of DNA after phleomycin treatment or EcoRI expression. Wild-type cells carrying pGAL-EcoRI (used for panel C) were grown in glucose and were synchronized at G₂/M phase with nocodazole. Cells were then either mock treated (−) or treated with 20 μg/ml phleomycin for 60 min (P). The same cells were grown in sucrose and were synchronized at G₂/M phase with nocodazole. After arrest, the culture was incubated with galactose to induce EcoRI expression for 120 min (E). Chromosomes were separated by pulsed-field gel electrophoresis, transferred to a nylon membrane, and hybridized with a probe to RDN5 genes on chromosome (Chr.) XII. (E) Effect of sae2Δ or mre11-3 mutation on accumulation of DNA-histone cross-links after phleomycin treatment. Wild-type, sae2Δ, and mre11-3 mutant cells were treated with phleomycin as for Fig. 1A. Extracts were separated on a CsCl gradient. Fractions containing chromosomal DNA were subjected to immunoblot analysis with an anti-histone H3 or anti-histone H2B antibody. The amount of DNA at the SMC2 locus was analyzed by real-time PCR and, for each fraction, was normalized to that from untreated wild-type cells. (F) Accumulation of DNA-histone cross-links after EcoRI induction. Wild-type and sae2Δ cells carrying pGAL-EcoRI (E) or the control vector (−) were treated as for panel C. As a positive control, sae2Δ mutants carrying the vector were grown in sucrose and were arrested with nocodazole at G₂/M. Cells were transferred to galactose medium and were either mock treated (−) or treated with 20 μg/ml phleomycin for 60 min (P). DNA-histone H3 cross-links were analyzed as for panel E. (G) Effect of mre11-3 mutation on Tel1 activation after phleomycin treatment. Wild-type and mre11-3 mutant cells expressing HA-tagged Tel1 were either mock treated (−) or treated with phleomycin (+) and were analyzed as for panel A.

In vitro reconstitution of Tel1 activation. (A) MRX- and DNA-dependent activation of Tel1 kinase. Tel1 or Tel1-KN protein was incubated with the MRX complex or a 150-bp DNA fragment (see Fig. 5A, N) in the presence of [³²P]ATP. GST-Rad53 was used as a substrate of protein kinase Tel1. Incorporation of ³²P into GST-Rad53 was detected by phosphorimaging. Phosphorylation levels of GST-Rad53 were normalized to that observed after incubation of wild-type Tel1 kinase without MRX or DNA. Relative phosphorylation was determined from three independent experiments. Bars represent averages; error bars, standard deviations. (B) DNA end-dependent Tel1 kinase activation. Tel1 and MRX were incubated with no DNA (−), the 150-bp DNA fragment (+), SmaI-digested pUC18 (D), or uncleaved pUC18 (U), and Tel1 activity was monitored as for panel A.

Effect of DNA end processing on protein kinase activity of Tel1 in vitro. (A) Degradation of 800-bp DNA fragments by the MRX complex. ³²P-labeled DNA fragments were incubated with MRX in the reaction buffer for 10 min. DNA species were analyzed as for Fig. 5B. The 150-bp fragment used here is DNA fragment N. The preparation of the 800-bp DNA fragment is described in Materials and Methods. (B) Effect of DNA length on Tel1 activation. Tel1 and MRX were incubated with a 150-bp or a 800-bp DNA fragment in the reaction buffer containing [³²P]ATP and GST-Rad53 for 10 min. The DNA fragments were the same as those used in panel A but were not ³²P labeled. The phosphorylation level of GST-Rad53 in each reaction product was normalized to that observed after incubation without DNA fragments. Relative phosphorylation was determined from three independent experiments. Bars represent averages; error bars, standard deviations. (C) Effect of mre11-3 mutation on MRX-dependent DNA degradation. The 150-bp ³²P-labeled DNA fragment N was incubated with or without the MRX or MRX^mre11-3 complex in the reaction buffer. DNA species were analyzed as for panel A. (D) Effect of mre11-3 mutation on Tel1 activation. Tel1 was incubated with a DNA fragment (N or 5′) and the MRX or MRX^mre11-3 complex in the reaction buffer containing [³²P]ATP and GST-Rad53 as for Fig. 4A. Fab-bound DNA fragments were prepared prior to the kinase reaction. The phosphorylation level of GST-Rad53 in each reaction was normalized to that observed with Tel1 after incubation with MRX and an unmodified DNA fragment. Relative phosphorylation was determined from three independent experiments. Bars represent averages; error bars, standard deviations.