pph3Δ ptc2Δ ptc3Δ cells are synergistically sensitive to DNA-damaging agents. (A) Wild-type and phosphatase deletion mutant cells were grown to the saturated phase in liquid YEP-dextrose medium. Ten-fold serial dilutions of the cultures were spotted on YEP-dextrose plates with or without DNA-damaging agents (0.02% methyl methanesulfonate [MMS], 50 mM hydroxyurea [HU], 1 μg/ml camptothecin [CPT]). To treat cells with UV, 10-fold serially diluted cells were spotted on YEP-dextrose plates, followed by irradiation with 50 J/m² UV. The photographs were taken 3 days after spotting. (B) After growth of wild-type or mutant cells to exponential phase in liquid culture, 0.05%, 0.1%, 0.15%, and 0.2% MMS were added into the culture. One hour after MMS treatment, the cells were washed with MMS-free medium three times and then plated on YEP-dextrose plates. Viable colonies were counted 3 days later. Percentage of viability was calculated by dividing the number of colonies from the MMS-treated culture by that from the untreated culture. Error bars were obtained from three different experiments. (C) Wild-type and phosphatase deletion mutant cells were grown in liquid YEP-dextrose medium to exponential phase and treated with or without 0.2% MMS for 1 h. Whole-cell extracts were subjected to Western analysis with either anti-γ-H2AX or anti-carboxypeptidase Y (CPY) antibody. (D) PPH3 is deleted either in wild-type or in h2a-S129A strains. The sensitivities to MMS were assessed by spotting 10-fold serially diluted cultures onto YEP-dextrose plates with or without 0.02% MMS. The photographs were taken 3 days after spotting.

SSA-mediated DSB repair is defective in pph3Δ ptc2Δ ptc3Δ cells. (A) Diagram showing the leu2-cs (centromere-proximal dark gray box), which contains an HO endonuclease cleavage site (pointed to with ▴) within the endogenous LEU2, and the U2 locus (centromere-distal dark gray box), which contains 3′ partial sequences of LEU2, on chromosome 3 in strain YMV80. The centromere is marked as a black dot. Vertical lines mark the recognitions sites of the Asp718 restriction enzyme, which was used for Southern analysis. Probe 1, marked as a black bar, is specific for the leu2-cs adjacent region that is centromere proximal. Probe 1 can detect three DNA fragments digested with Asp718, which are denoted as “uncut,” “cut,” and “repair.” (B) Either pph3Δ ptc2Δ ptc3Δ or wild-type cells were collected 1, 2, 4, 8, 12, and 24 h after as well as prior to HO induction. Genomic DNA purified from the cells was digested with an Asp718 restriction enzyme and subjected to Southern analysis using probe 1. (C) PCR-based nonhomologous (NH) tail removal assay. The diagram in the upper panels shows an intermediate step of SSA-mediated DSB repair in strain YMV80. The primers used for the NH tail cleavage assay are denoted as p1 and p2, which are specific to the leu2-cs adjacent region that is centromere distal and to the leu2-cs, respectively. The annealed homologous sequences are marked in gray boxes. The lower panel shows quantified PCR results using the p1/p2 primer pair. The PCR product by the p1/p2 primer was run on 1.5% agarose gel, and then the intensities of PCR product on the gel were measured by Quantity-One (Bio-Rad). Then the signal was normalized by the PCR signal with the CEN8 p1/p2 primer pair used as a loading control. The centromere 8 (CEN8)-specific region is amplified by the pair of CEN8 p1/p2 primers. The normalized PCR ratio before HO induction was set as 100%. (D) PCR-based primer extension assay. The upper panel shows a diagram demonstrating an intermediate step of SSA-mediated DSB repair in the strain YMV80. Newly synthesized donor-specific sequences are shown as waves, and the homologous regions are in gray. p3 and p4 denote primers specific to the donor and to the leu2-cs adjacent region that is centromere proximal, respectively. The lower panels show representatives of the PCR results obtained by using the p3/p4 pair as well as the CEN8 p1/p2 pair (loading control). (E) Quantification of PCR product shown in panel D. The band intensities on 1.5% agarose gel were measured by Quantity-One (Bio-Rad). The ratio of the p3/p4 PCR product to the loading control (PCR product by CEN8 p1/p2) at 24 h after HO induction in wild-type cells was referred to as 100%. Subsequently, the quantified PCR values at all time points were converted to percentages. The error bar was obtained from three different PCRs.

Intrachromosomal GC-mediated DSB repair is moderately defective in pph3Δ ptc2Δ ptc3Δ cells. (A, panel a) Diagram of MATa and HMLα-inc on Chr3. The vertical lines indicate StyI cleavage sites. ▴ marks an HO-induced DSB. The black bar denotes probe 3, specific to the MAT region, which can detect “MATa” fragment prior to HO induction. Probe 3 can detect the “HO cut,” “repair (MATα-inc),” and “MAT distal” fragments. Because of the sequence homology to HMLα-inc, probe 3 also detects “donor (HMLα-inc).” The centromere is marked as a black dot. (b) Diagram showing an intermediate step of intrachromosomal MAT switching. Newly synthesized DNA specific to HMLα-inc sequences is shown as waves. p7 and p8 mark the primers used for the PCR-based primer extension assay in panel D. (B) Southern analysis showing intrachromosomal MAT switching at 30, 45, 60, 90, 120, 150, 300 min after as well as prior to HO induction. *, nonspecific bands detected by probe 3. (C) Percentage values of repair products measured in panel B. The calculation was done as described in the legend to Fig. 5C. (D) Percentage values of the PCR-based primer extension assay during intrachromosomal MAT switching with the p7/p8 primer pair. The calculation was done as described in the legend to Fig. 4E.

Strains used to study the roles of Pph3, Ptc2, and Ptc3 in HR-mediated DSB repair. (A, panel a) Strain YJK139 contains the endogenous HMLα-inc locus but lacks the HMRa locus. An HO-induced DSB at MAT on chromsome 3 (Chr3) is repaired by intrachromosomal gene conversion (Intra-GC) using HMLα-inc on the same chromosome as a donor. The centromere is marked as a black dot. (b) Both endogenous HML and HMR loci are deleted in strain YJK17. An HO-induced DSB at MAT on Chr3 is repaired by interchromosomal gene conversion by copying the MATa-inc sequence on chromosome 5 (Chr5). (B) Strain YMV80 contains an HO cleavage site within the LEU2 sequence (leu2-cs) on Chr3. The 3′ partial sequence of LEU2 (U2) is located 30 kb apart from the leu2-cs. An HO-induced DSB on the leu2-cs is repaired by single-strand annealing (SSA) using the U2 sequence as a homologous donor. The centromere is marked as a black dot.

Triple deletion of PPH3, PTC2, and PTC3 reduces the viability of cells after the induction of a DSB by HO endonuclease. (A) Viabilities of the YMV80 wild-type strain and its derivatives lacking Pph3, Ptc2, or Pph3 on galactose plates. Wild-type and mutant cells were grown in liquid YEP-lactate culture to exponential phase, and then cells from each culture were plated either on YEP-dextrose or YEP-galactose plates. Three days after plating, the number of colonies on each plate was counted. The percentages of viability of the wild type and each mutant were calculated by dividing the number of colonies on the galactose plates by that on the dextrose plates. The graph shows an average of three different experiments. *, result significantly different from that for wild-type cells; **, significantly different viability compared to that of ptc2Δ ptc3Δ cells. (B) Percentages of viability of the YJK17 wild-type strain and its derivatives lacking Pph3, Ptc2, or Pph3 on galactose plates. Experiments were performed as described for panel A. *, result significantly different from that for wild-type cells.

Interchromosomal ectopic GC-mediated DSB repair is defective in pph3Δ ptc2Δ ptc3Δ cells. (A) Diagram of MATα in Chr3 and MATa-inc in Chr5. The vertical lines indicate EcoRI cleavage sites. ▴ marks an HO-induced DSB. The black bar denotes probe 2, specific to the MAT region, which can detect a “MAT” fragment either prior to HO induction or when the DSB is repaired. Probe 2 can also detect the “HO cut” as well as “MATa-inc” on Chr5. (B) Exponentially growing wild-type and mutant cells were collected at different time points after as well as prior to the induction of HO. Genomic DNA from the cells at each time point was digested with EcoRI and then run on a 0.8% agarose gel for Southern analysis. (C) The percentage of value of the repair product at each time point was quantified by Quantity-One (Bio-Rad). The ratio of “MAT” to “MATa-inc” was calculated at each time point. The ratio was then was normalized with that prior to HO induction, which was referred to as 100%. (D) Diagram showing the formation of a synapsis between 3′-tailed single-strand DNA from Chr3 and the homologous duplex region in Chr5. Newly synthesized DNA specific to MATa-inc sequence in Chr5 is shown as waves. “a” denotes the region amplified by PCR following Rad51 ChIP, which is about 200 bp away form the HO-induced DSB. “b” indicates the Chr3-specific region, ∼500 bp distal to the HO-induced DSB. “c” is the Chr5-specific region. (E) Rad51 ChIP values at single-stranded regions (a and b) on Chr3 as well as a homologous donor region on Chr5 (c). The fold increase was measured by normalizing the Rad51 ChIP signal at each time point after HO induction with that prior to HO induction. (F) Representative results from PCR assays to show primer extension from the invading strand. Genomic DNA either from wild-type cells or from pph3Δ ptc2Δ ptc3Δ cells, which was purified for Southern analysis in Fig. 5B, was subjected to PCR using the p5 and p6 primers in panel D. As a loading control, the CEN8-specific sequences were amplified by CEN8 p1/p2 primers. (G) Percent value of PCR-based primer extension assay. The calculation was done as described in the legend to Fig. 4E.