The lack of Tel1 impairs Rad53 phosphorylation and single-stranded DNA formation in response to a single DNA double-strand break. (A) YEP+raf nocodazole-arrested cell cultures of wild-type (WT) JKM139 and isogenic tel1Δ, exo1Δ and tel1Δ exo1Δ strains (time 0) were transferred to YEP+raf+gal to induce HO expression in the presence of nocodazole. Western blots of protein extracts prepared at the indicated time points were probed with Rad53 antibodies. (B) Schematic representation of the region immediately centromere-distal to the MAT HO site (bottom), and of the DSB and 5′-to-3′ resection products (top) detectable with the indicated ssRNA probe after alkaline gel electrophoresis of SspI (S)-digested DNA. The probe is specific for the MAT locus and reveals a 1.1-kb fragment from the uncut MAT locus. When HO cuts the MAT locus, a smaller 0.9-kb HO-cut fragment is produced. 5′-to-3′ resection progressively eliminates SspI sites, generating larger ssDNA SspI fragments (r1–r7) detected by the probe. (C) Genomic DNA prepared from samples taken at the indicated time points during the experiment in (A) was digested with SspI and run on alkaline agarose gel, followed by gel blotting and hybridization with the ssRNA probe shown in (B). DSB, DNA double-strand break; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA; YEP+raf, yeast extract peptone and raffinose; YEP+raf+gal, YEP+raf and galactose.

EXO1 overexpression overcomes the defective response of tel1Δ and mre11Δ cells to a single DNA double-strand break. (A,B) YEP+raf nocodazole-arrested cell cultures of wild-type (WT) JKM139 and isogenic mre11Δ and tel1Δ strains containing 2μ plasmids, either empty or carrying the EXO1 gene (time 0), were transferred to YEP+raf+gal in the presence of nocodazole to induce HO expression. Genomic DNA from samples collected at the indicated time points was analysed as described in Fig 1B. (C) Western blots of protein extracts prepared at the indicated times were probed with Rad53 antibodies. DSB, DNA double-strand break; Exp, exponentially growing cells; YEP+raf, yeast extract peptone and raffinose; YEP+raf+gal, YEP+raf and galactose.

Response to a single DNA double-strand break in mec1Δ cells. (A,B) Cell cultures of wild-type (WT) JKM139 and isogenic exo1Δ, mec1Δ and mec1Δ exo1Δ strains, exponentially growing in YEP+raf (time 0), were transferred to YEP+raf+gal to induce HO expression. Samples withdrawn at the indicated times were used for FACS analysis of DNA contents (A) and western blot analysis of protein extracts with Rad53 antibodies (B). (C,D) Cell cultures of wild-type JKM139 and isogenic GAL-TEL1, GAL-TEL1 mec1Δ and mec1Δ strains, exponentially growing in YEP+raf (time 0), were transferred to YEP+raf+gal to induce HO expression. Samples were collected at the indicated times for western blot analysis with Rad53 antibodies (C) and to determine the percentage of mononucleate large budded cells (D). DSB, DNA double-strand break; FACS, fluorescence-activated cell sorting; YEP+raf, yeast extract peptone and raffinose; YEP+raf+gal, YEP+raf and galactose.

Multiple DNA double-strand breaks trigger Tel1-dependent checkpoint activation. (A,B) Cell cultures of wild-type (WT) LSY1170, LSY1223, LSY1259 and isogenic mec1Δ strains, exponentially growing in raffinose-containing selective medium (time 0), were transferred to YEP+raf+gal to induce HO expression. Samples collected at the indicated times were used to determine the percentage of mononucleate large budded cells (A) and for western blot analysis with Rad53 antibodies (B). (C) LSY1259 mec1Δ and isogenic mec1Δ tel1Δ cell cultures, exponentially growing in raffinose-containing selective medium (time 0), were transferred to YEP+raf+gal to induce HO expression, and protein extracts prepared at the indicated times were used for western blot analysis with Rad53 antibodies. (D) Cell cultures of wild-type LSY1170, LSY1259 and isogenic mec1Δ strains, all expressing the MRE11-HA3-tagged allele from the corresponding endogenous promoter and exponentially growing in raffinose-containing selective medium (time 0), were transferred to YEP+raf+gal to induce HO expression. Samples withdrawn at the indicated times were used for western blot analysis with HA antibodies. (E) Nocodazole-arrested (noc) cell cultures of wild-type JKM139 and isogenic exo1Δ, mec1Δ, mec1Δ exo1Δ, tel1Δ, tel1Δ exo1Δ and tel1Δ mec1Δ strains were transferred to YEPD containing 10 μg/ml phleomycin and 15 μg/ml nocodazole (+phleo +noc). Western blot analysis with Rad53 antibodies was carried out on protein extracts prepared at the indicated times. (F) α-factor-arrested wild-type JKM139 and isogenic mec1Δ, tel1Δ and tel1Δ mec1Δ cell cultures were transferred at time zero (αf) in YEPD containing 5 μg/ml phleomycin and 5 μg/ml α-factor (+phleo +α-factor), followed by western blot analysis with Rad53 antibodies on protein extracts prepared at the indicated times. Exp, exponentially growing cells; YEP+raf, yeast extract peptone and raffinose; YEP+raf+gal, YEP+raf and galactose.

Kinetics of DNA double-strand break resection and Tel1-dependent checkpoint activation. (A–C) Cell cultures of LSY1259 (WT) and isogenic mec1Δ, exo1Δ and mec1Δ exo1Δ strains, exponentially growing in raffinose-containing selective medium (time 0), were transferred to YEP+raf+gal to induce HO expression. Samples were collected at the indicated times to prepare genomic DNA for the analysis of DSB resection at the MATα locus (A), protein extracts for western blot analysis with Rad53 antibodies (B) and to determine the percentage of mononucleate large budded cells (C). To detect DSB formation and 5′-to-3′ resection products, genomic DNA was digested with BamHI and StyI and separated on alkaline agarose gel. As indicated on the right part of (A), gel blot hybridization with the indicated single-stranded RNA probe specific for the MAT locus reveals HO-cut and uncut fragments of 0.7 and 1.9- kb, respectively. 5′-to-3′ resection progressively eliminates BamHI (B) and StyI (S) sites, generating larger ssDNA fragments (r1–r6) detected by the probe. (D, E) Cell cultures of LSY1259 mec1Δ strains transformed with 2μ plasmids, either empty or carrying the EXO1 gene, exponentially growing in raffinose-containing selective medium (time 0), were transferred to YEP+raf+gal to induce HO expression. Samples were collected at the indicated times to prepare genomic DNA (D) for the analysis of DSB resection as in (A) and protein extracts for western analysis with Rad53 antibodies (E). DSB, DNA double-strand break; YEP+raf, yeast extract peptone and raffinose; YEP+raf+gal, YEP+raf and galactose.