(A–D) Anchoring away Ctf19 after DNA replication and cohesion establishment is not sufficient to prevent the appearance of centromere-proximal DSBs. (A, B) Scheme of the anchor away system and experimental setup used to deplete Ctf19 during meiosis. (C) Addition of Rapamycin leads to Ctf19 removal from the pericentromere. Three cultures of strain AM18978 (CTF19-FRB-GFP RPL13A-2XFKBP12 tor1-1 fpr1△ ndt80△ REC8-3HA) were induced to sporulate. Either DMSO or Rapamycin were added to two of the cultures (t=0). Rapamycin was added 3 hr after inducing sporulation to the third culture (t=3). A fourth culture was a no tag ndt80△ control (AM11633) to which DMSO was added. All cultures were harvested 5 hr after inducing sporulation (prophase I arrest) and Ctf19 levels were analyzed by anti-GFP ChIP-qPCR at the indicated sites. Error bars represent standard error (n=4 biological replicates). *p<0.05, paired t-test. See Figure 5 and Supplementary file 4B for details of primer sets used. (D–F) Anchoring away Ctf19 after DNA replication allows establishment of centromeric Rec8. (D) Cells treated as in (C) were processed for anti-HA ChIP-qPCR at the indicated sites. Error bars represent standard error (n=4 biological replicates). *p<0.05, paired t-test. (E and F) Three cultures of strain AM20138 (CTF19-FRB-GFP NDC10-6HA pGAL-NDT80 pGPD1-GAL4(848)-ER RPL13A-2XFKBP12 tor1-1 fpr1△ ndt80△ REC8-3HA) were resuspended in sporulation medium (t=0). DMSO or Rapamycin were immediately added to the first and second cultures (t=0), while the third culture received Rapamycin after 3 hr incubation (t=3). After 6 hr total, β-estradiol was added to release cells from the prophase I arrest, samples were harvested at 15 min intervals and chromosome spreads were prepared and stained with anti-HA and anti-Myc antibodies. (E) Examples of binucleate cells with centromeric or no Rec8. (F) Percentages of binucleate cells with centromeric Rec8 are shown for indicated conditions. (G–I) Timed depletion of Ctf19 to test DSB formation dependencies. (G) DNA replication is largely complete prior to anchoring Ctf19 away in cultures where Rapamycin was added at 3 hr. A control strain GV2367 (RPL13A-2XFKBP12 tor1-1 fpr1△ dmc1△) and equivalent experimental strain carrying CTF19-FRB-GFP (GV2354) were induced to undergo meiosis together with a ctf19△ dmc1△ mutant (GV1912). Rapamycin or DMSO were added at the indicated times (red circles, addition of DMSO at t=0; blue squares, addition of Rapamycin at t=0; green squares, addition of Rapamycin at t=3) and samples were processed for FACS analysis (timepoints t=0, 3, 5, 8 hr). (H) Analysis of DSB formation in the experiment shown in (B). Southern blot shows that DSB formation close to CEN1 occurs either when Ctf19 is anchored away early (t=0) or after DNA replication and cohesin enrichment (t=3). Red circles, addition of DMSO at t=0; blue squares, addition of Rapamycin at t=0; green squares, addition of Rapamycin at t=3; Arrowheads, Spo11-dependent DSBs; asterisks, cross-hybridizing species. (I) Quantification DSBs shown in (H). (J, K) Inhibition of centromere-proximal DSBs does not depend on cohesin. (J) CEN1-proximal DSBs are observed in rec8△ cells only upon deletion of Ctf19 complex components. Strains GV48 (dmc1△), GV2050 (dmc1△ mcm21△), GV2403 (dmc1△ rec8△), GV2286 (dmc1△ mcm21△ rec8△) were analyzed by Southern blotting as described in Figure 4B. (K) Cohesin impairment does not allow CEN1-proximal DSB formation. Strains used were GV48 (dmc1△), GV1912 (dmc1△ ctf19△), GV2305 (dmc1△ SCC4) and GV2533 (dmc1△ scc4-m35). (L) Anchoring away Ctf19-FRB after DNA replication and cohesin establishment leads to only a modest increase in pericentromeric COs. Three cultures of strain AM19543 [carrying heterozygous pericentromeric RFP/GFP reporters separated by ~10 kb, homozygous chromosomal arm CFP reporters (Figure 1B), estradiol-inducible Ndt80 to allow release from prophase I arrest (pGAL-NDT80 pGPD1-GAL4(848)-ER) and the Ctf19 anchor away system (CTF19-FRB RPL13A-2XFKBP12 tor1-1 fpr1△)] were resuspended in sporulation medium (t=0). DMSO or Rapamycin were immediately added to the first and second cultures (t=0), while the third culture received Rapamycin after 3 hr incubation (t=3). All cultures were incubated for 6 hr total before being released from the prophase I arrest by addition of β-estradiol and tetrads were scored after incubation for 48 hr total. ChIP, chromatin immunoprecipitation; DMSO, dimethyl sulfoxide; DSBs, double strand breaks; FACS, fluorescence-activated cell sorting; qPCR, quantitative polymerase chain reaction.