Resection in late mitosis is almost as efficient as in G2/M.

(a) Schematic representation of the HO resection assay. Primers (blue arrows) are designed to amplify a sequence that contains a StyI target site adjacent to the HO cutting site (HOcs). When this restriction enzyme is used on the extracted genomic DNA, amplification is inhibited. If resection extends beyond the StyI site, StyI does not cut and the primers can amplify. (b) Summary table of the amplification yield obtained after the StyI digestion for each situation (mock control vs. HO induction). (c) Charts depicting the resection kinetics for two different amplicons located at 726 bp and 5.7 kb downstream the HOcs break (mean ± sem, n=3; fresected is the proportion of resected DNA. (d) Representative Western blots against Rad53 to follow the sensing of DNA damage detection in G2/M versus late mitosis.

Yeast cells use HR to repair the HO DSB in late mitosis.

(a) Schematic of the fragments obtained after a StyI digestion for both MATa and MATα sequences. The probe to detect the fragments by Southern blot is in blue. When the MATa locus is intact, the digestion gives rise to a 0.9 kb fragment. The HO cutting site (HOcs) is located within the StyI-digested MATa locus. Thus, the HO-driven DSB shortens the fragment to 0.7 kb. HR leads to a gene conversion to MATα, which results in the loss of a StyI restriction site and a new fragment of 1.8 kb. (b-d) Cells were first blocked in the cdc15-2 arrest at 34 ºC for 3 hours (Tel). Then, the HO-mediated DSB was generated by adding 2 µM β-estradiol. After 1 hour, the β-estradiol was washed away, and samples were taken to monitor the repair for 3 hours. (b) Representative Western blot analyses for HO induction and subsequent degradation (tagged with Flag epitope) and the DSB sensing through Rad53 hyperphosphorylation. A PGK1 western blot served as a housekeeping and Ponceau-S staining of the membrane as a loading control for all lanes. The leftmost lane in the Ponceau-S corresponds to the protein weight marker. (c) Representative Southern blot for the MAT switching assay in late mitosis. Alongside the MAT probe, a second probe against the ACT1 gene (1.1 kb fragment) was included for normalization. The MAT probe also recognizes an allele-independent MAT distal fragment (2.2 kb). (d) Quantification of relative band intensities in the MAT switching Southern blots (mean ± sem, n=3). Individual values were normalized to the ACT1 signals. Then, every lane was normalized to MATa at the arrest. Tel: Telophase. +β-E: β-estradiol addition.

Smc3 degradation delays HR at the MAT locus in late mitosis.

(a) Serial dilution spot assay for the SMC3:AID* derivative of the strain used in Figure 2. The effect of Smc3-aid* degradation was tested in the presence of 8 mM IAA. The same strain without the ubiquitin-ligase OsTIR1 was also used as the control. (b-d) Cells were treated as in Figure 2 but including a 1 h IAA step between the cdc15-2 arrest and the induction of HO. IAA was then maintained throughout the experiment. (b) Representative Western blot as in Figure 2b but also including Smc3-aid* decline after IAA addition. (b) Representative Southern blot for the MAT switching assay after degrading Smc3-aid* in late mitosis. (d) Quantification of relative band intensities for MAT switching Southern blots (mean ± sem, n=3). Values were normalized as in Figure 2d. The MAT switching in the wild type strain is represented alongside for comparison. Tel: Telophase. +β-E: β-estradiol addition. +IAA: Indole-acetic acid.

Scc1 returns after DSBs in late mitosis and the absence of Smc3 inhibits sister telomere coalescence.

(a) Western blot against Scc1-3myc (detected with an α-myc antibody). This experiment compares Scc1-3myc levels in an asynchronous, G2/M- and telophase-blocked cultures. Cells arrested in telophase were further subdivided into three subcultures and treated as indicated for another two hours (mock, phleomycin, and HO endonuclease). The leftmost lane is a control strain for Scc1 without the 3myc epitope tag. PGK1 protein served as a housekeeping. Ponceau-S staining is also shown as a loading control. Asyn.: Asynchronous. Tel: Telophase. +Phle: 10 mg·mL-1 phleomycin. +HO: 2 μM β-estradiol. *: Unspecific band detected by the α-myc antibody just over the Scc1-3myc signal. (b, c) A SMC3:AID* strain that reports the segregation status of the chromosome XII right telomere (cXIIr-Tel) was used for this regressed anaphase assay. Cells arrested in late mitosis were divided into two. One subculture was treated with 8 mM IAA for 1 h. Then, each subculture was, in turn, divided into two, one serving as a mock control and the other was treated with 10 μg·mL-1 phleomycin for an extra hour. Samples were taken at the indicated time points for Western blotting and microscopy. (b) Representative Western blot. Smc3-aid* was monitored using an anti-mini-AID monoclonal antibody. Rad53 hyperphosphorylation was also monitored as a control to confirm DNA damage after phleomycin addition. PGK1 was used as the housekeeping control to quantify the remaining Smc3 signal. Ponceau-S staining of the membrane is also shown as a loading reference for each lane. (c) Chart representing quantifications of sister telomere segregation status (mean ± sem, n=3). Cells were classified into three categories depending on cXIIr-Tel location within the elongated nucleus: (i) Segregated, showing two foci well separated in distinct cell bodies; (ii) same body, presenting both sister telomeres in one of the daughter-to-be cells; and (iii) coalescence, only one cXIIr-Tel focus. Statistical significance was calculated through 2-way ANOVA and multiple comparisons analysis. IAA: Indole-acetic acid; Tel: Telophase (i.e. late mitosis); ns: non-significant statistical difference.

Strains used in this work.