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. 2022 Sep 20;11:e78015. doi: 10.7554/eLife.78015

Figure 3. Proximal mobility is Rad9 independent.

(A) Non-repaired cells (white cells, n=584) show increased global mobility, while repaired cells (red cells, n=519) recover normal global mobility. Mean squared displacements (MSDs) of the visualization of the locus on chromosome V (V-Vis) locus in strain C as a function of time, calculated as in Figure 2A. The gray and red curves correspond respectively to strain C in the presence of the donor dCen plasmid after 12 hr in galactose medium with a plasmid not expressing I-SceI (-DSB) and expressing I-SceI (+DSB). MSDs are calculated after color visual cell sorting. Red MSD full curve corresponds to cycling red cells, red empty curve corresponds to white G2/M arrested cells. Examples of white, G2/M arrested and red, cycling cells are shown. Bar scale, 2 µm. Seven independent experiments were done. (B) Box plots of MSDs at 10 s of undamaged, white and red cells 12 hr after I-SceI induction. The color code corresponds to that used in (A). (C) MSDs in strains C and S in wild-type (WT) and Δrad9 mutant. MSDs are calculated as in Figure 2 upon 6 hr of double-strand break (DSB) induction with empty plasmid or with dCen plasmid (+DSB: n=380 [C strain], n=103 [S strain], and +DSB+dCen: n=452 [C strain], n=98 [S strain], respectively). Controls without DSB are also shown (-DSB: n=590 [C strain], n=176 [S strain]). (D) Homologous recombination (HR) kinetics upon induction of I-SceI in the presence of dCen in WT (black) and Δrad9 (orange) C or S strains were measured by FACS as in Figure 1F. Error bars represent the standard deviation of three independent experiments.

Figure 3—source data 1. Mean squared displacement (MSD) values.
Figure 3—source data 2. Mean squared displacement (MSD) values at 10 s.
Figure 3—source data 3. Mean squared displacement (MSD) values.
Figure 3—source data 4. Values for repaired red cells.

Figure 3.

Figure 3—figure supplement 1. Implication of Rad9 in survival, cell cycle and global dynamics.

Figure 3—figure supplement 1.

(A) Scatter plot showing the mean squared displacement (MSD) at 10 s of the damaged-only population (in white), the repaired-only population (in red), or the 20–80% mixed population (in gray) 6 or 12 hr after double-strand break (DSB) induction.( B) I-SceI cleavage efficiency measured in Δrad9 mutant by qPCR using primers flanking the I-SceI cut site (cs). The error bars represent the standard deviation of three independent experiments. (C) Left: Analysis of the cell cycle of Δrad9 C and S strains after induction of I-SceI with dCen. Quantification is done as in Figure 1D. The peaks corresponding to 1n and 2n amount of DNA are indicated. Right: Drop assay (10-fold serial dilutions) showing the sensitivity of wild-type (WT) and Δrad9 C or S strains, containing a plasmid expressing or not I-SceI (-DSB, +DSB respectively), with dCen or an empty plasmid, when I-SceI is induced for 48 hr (galactose, Gal) or not induced (raffinose, Raff). (D) Boxplots of the distribution of MSDs at 10 s from Figure 3C for WT and Δrad9 C and S strains. Median values, lower and upper quartiles are shown. Whiskers indicate the full range of measured values, except for outliers represented by small red dots. Parentheses indicate the result of a Wilcoxon rank-sum test between distributions, with the p-value. n.s., non significant, * (p<0.05), ** (p<0.001), *** (p<0.0001).
Figure 3—figure supplement 1—source data 1. qPCR values for I-SceI efficiency.
Figure 3—figure supplement 1—source data 2. Spot assay for cell survival to I-SceI induction.
Figure 3—figure supplement 1—source data 3. FACS values for cell cycle analysis.
Figure 3—figure supplement 1—source data 4. Mean squared displacement (MSD) values at 10 s.