Fig. 9: The effect of NPFs on DSB repair by gene conversion. | Nature Communications

Fig. 9: The effect of NPFs on DSB repair by gene conversion.

From: Arp2/3 and type-I myosins control chromosome mobility and end-resection at double-strand breaks in S. cerevisiae

Fig. 9

A Repair strain YJK17. The donor MATa-inc on chromosome 5 is used as a donor to repair the Gal-HO-cut in the MAT locus on chromosome 3 through gene conversion (GC). B Survival assay in YJK17 mutants: YJK17 (WT) (n = 6), FZ063 (myo3Δ) (n = 3), FZ040 (myo5Δ) (n = 3), FZ177 (Las17-AID) (n = 3), Las17-AID + IAA (1 mM) (n = 3), FZ172 (mre11Δ) (n = 3), FZ193 (Las17-AID mre11Δ) (n = 6), and Las17-AID mre11Δ + IAA (n = 6). ~200–100 cells from a YP-Lac culture were plated on YPD, YP-Gal, and YP-Gal+IAA plates. The survival rate was calculated by the number of cells grown on a YEP-Gal or YEP-Gal+IAA plate divided by the number of cells grown on a YPD plate. Statistical analysis for the percentage of survivors was derived in PRISM using a one-way Anova (ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001). WT vs. myo3Δ (p = 0.6053), WT vs. myo5Δ, (p = 0.6505), WT vs. mre11Δ, (p = 0.3879), Las17-AID vs. Las17-AID + IAA (p < 0.0001), and Las17-AID mre11Δ vs. Las17-AID mre11Δ + IAA (p < 0.0001). See Supplementary Data 3 for significance. See “Methods” section for more details. Mean ± SEM is shown. Source data are provided as a Source Data file. C Survival assay of YJK17 (n = 6) and YJK17 + nocodazole (Noc) (10 µg/ml) (n = 6). Noc was added 1 h before galactose. See “Methods” section for more details. Mean ± SEM is shown. Source data are provided as a Source Data file. D Survival assay of YJK17 mutants normalized to wildtype + nocodazole (10 µg/ml): YJK17 + Noc (WT + Noc) (n = 6), Las17-AID + Noc (n = 6), Las17-AID + IAA (n = 6), Las17-AID + IAA + Noc (n = 6), Las17-AID mre11Δ (n = 6), Las17-AID mre11Δ + IAA (n = 6), and Las17-AID mre11Δ + IAA + Noc (n = 6). Survival assay conducted as described in (B). Nocodazole (10 µg/ml) was added 1 h before galactose. IAA (1 mM) was added 1 h before galactose. See “Methods” section for more details. Statistical analysis for the percentage of survivors was derived in PRISM using a one-way Anova (ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001). Las17-AID + Nocodazole vs. Las17-AID + IAA (p = 0.0067), Las17-AID + IAA vs. Las17-AID + IAA Nocodazole (p = 0.0038), Las17-AID + Nocodazole vs. Las17-AID + IAA + Nocodazole (p = 0.8219), Las17-AID mre11Δ + Nocodazole vs. Las17-AID mre11Δ + IAA (p = 0.0001), Las17-AID mre11Δ + IAA vs. Las17-AID mre11Δ + IAA Nocodazole (p = 0.0007), and Las17-AID mre11Δ + Nocodazole vs. Las17-AID mre11Δ + IAA + Nocodazole (p = 0.5676). See Supplementary Data 3 for significance. Mean ± SEM is shown. Source data are provided as a Source Data file. E Mating-type switch strain MY270. The donor HMLα-inc is used as a donor to repair the Gal-HO cut in the MAT locus on chromosome 3 through gene conversion (GC). After repair, MATα-inc cannot be cut by Gal-HO78. F Survival assay of MY270 (n = 6) and MYO270 + CK-666 (100 μM) (n = 6). Survival assay conducted as described in (B). CK-666 (100 µM) or 200 proof ethanol was added 20 min before plating on YPD and YP-Gal plates. Statistical analysis for the percentage of survivors was derived in PRISM using a two-sided unpaired t-test (ns p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001). WT vs. WT + CK-666 (p = 0.7417). See Supplementary Data 3 for significance. Mean ± SEM is shown. Source data are provided as a Source Data file. G Repair strain YMV2 and repair by single-strand annealing (SSA) or break-induced replication (BIR). An HO cut site is inserted in LEU2 on chromosome 3 (LEU2::HOcs), and a 1.3 kb donor of the C-terminal end of LEU2 (U2) is inserted 30 kb upstream of LEU2::HOcs. The 30 kb sequence between the homologous sequences is deleted after repair by either SSA or BIR. H Survival assay of YMV2 strains grown on YP-Gal-IAA plates: YMV2 (WT) (n = 3), YMA65 (rad51Δ) (n = 3), YMA63 (Las17-AID) (n = 3), YMA63 + IAA (Las17-AID + IAA) (n = 3), YMA66 (rad51Δ Las17-AID) (n = 3), and YMA66 + IAA (rad51Δ Las17-AID + IAA) (n = 3). Survival assay conducted as described in (B). WT vs. rad51Δ (p > 0.9999), WT vs. Las17-AID (p = 0.9807), WT vs. rad51Δ Las17-AID (p = −0.9807), WT vs. Las17-AID + IAA (p = 0.0172), WT vs. rad51Δ Las17-AID + IAA (p < 0.0001), Las17-AID vs. Las17-AID + IAA (p = 0.0038), and rad51Δ Las17-AID vs. rad51Δ Las17-AID + IAA (p < 0.0001). See Supplementary Data 3 for significance. Mean ± SEM is shown. Source data are provided as a Source Data file.

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