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

Figure 1. Tracks homologous recombination in vivo (THRIV), a system to follow homologous recombination (HR) in vivo.

(A) Schematic representation of the two sequences used to track recombination events during time. The recipient mCherry-I-SceI has a 30pb I-SceI sequence followed by a stop codon (red bar) inserted at 57 amino acids from the end of the mCherry coding sequence. The donor (dCen) is a full-length mCherry lacking promoter and terminator sequences, carried on a centromeric plasmid. A functional mCherry could be expressed only after HR. (B) Schematic 2D representation of the Rabl spatial configuration of two chromosomes in the nucleus of Saccharomyces cerevisiae. The spindle pole body (rectangle), the microtubules (lines), and the centromeres (spheres) are in yellow. Telomeres are shown as gray/black spheres attached to the nuclear envelope. An orange circle represents the dCen plasmid. The gray crescent symbolizes the nucleolus. Lightening symbolize the targeted I-SceI cutting sites (cs) on chromosome IVR arm, with same color code as in the linear representation represented below. The cs are respectively located at 5 kb from centromere CEN IV (red, strain C), 400 kb from CEN IV (green, strain L), and 20 kb from telomere TEL IVR (blue, strain S), distances are in kb The green dot visualizes the locus used to monitor mobility on undamaged chromosome VR. (C) I-SceI cleavage efficiency is measured in a non-donor strain by qPCR using primers flanking the I-SceI cs. The error bars represent the standard deviation of five or two independent experiments for strain C and strains L, S, respectively. (D) FACS analysis of the cell cycle of strains C, L, and S after induction of I-SceI with or without dCen plasmid. For the quantification of the cell cycle progression, the amount of DNA is measured with Sytox after fixation with ethanol and treatment with RNase. The peaks corresponding to 1n and 2n amount of DNA are indicated. (E) Drop assay (10-fold serial dilutions) showing the sensitivity of C, L, and S strains, containing a plasmid expressing or not I-SceI (-DSB, +DSB, respectively), with dCen plasmid or without (empty plasmid), when I-SceI is induced for 48 hr (galactose) or not (raffinose). (F) HR kinetics upon induction of I-SceI in the presence of dCen in C, L, or S strains. Percentages of repaired red cells were measured by FACS after PFA fixation in the absence of Sytox. Error bars represent the standard deviation of three independent experiments, five independent experiments for strain C. Mann-Whitney test, n.s., non-significant.

Figure 1—source data 1. qPCR values for I-SceI efficiency.
Figure 1—source data 2. FACS values for cell cycle analysis.
Figure 1—source data 3. Spot assay for cell survival to I-SceI induction.
Figure 1—source data 4. FACS values for repaired red cells.

Figure 1.

Figure 1—figure supplement 1. Validation of the THRIV system.

Figure 1—figure supplement 1.

(A) Growth curve of strains C (red), L (green), and S (blue) showing a similar doubling time. The error bars represent the standard deviation of three independent experiments. (B) I-SceI induction ratio measured by transcription quantitative reverse PCR (RT-qPCR) using primers hybridizing into the I-SceI coding sequence. The error bars represent the standard deviation of three independent experiments, except for 6 hr done two times. (C) Quantification of red and white cells in strains C and S, 48 hr after I-SceI induction on galactose plates before DNA extraction and PCR amplification around the I-SceI cut site (cs) of those strains. The error bars represent the standard deviation of two independent experiments, the total amount of counted cells was around 200/experiment. If the recipient I-SceI cs containing sequence is fully repaired by homologous recombination with the donor dCen plasmid, it cannot be cut by I-SceI in vitro. pGPS2 I-SceI cs containing plasmid is used as a control for in vitro digestion. (D) Boxplot of 2D distances measured in μm between the spindle pole body (SPB) (SPC29-mCherry, red) and dCen plasmid containing tetO repeats, labeled in green (TetR-GFP). As references, chromosome loci labeled either close to the centromere (YDR003w, 5 kb from CenIV) or the telomere (YER188C, 8 kb from TELVR) are shown. Between n=169, 3520 and 3461 cells were analyzed for each strain in three independent experiments. Brackets indicate the result of a rank-sum test between distributions, with ‘n.s.’ for ‘not significant’ (p>0.05) and *** for p<10–3. Below, examples of cell nuclei with the two red and green labeled loci allowing calculation of the SPB-loci distances. As expected, the donor sequence dCen on the centromeric plasmid is located close to the SPB, the subtelomeric sequence far from it, see Therizols et al., 2010.
Figure 1—figure supplement 1—source data 1. DO values for growth curve.
Figure 1—figure supplement 1—source data 2. Transcription quantitative reverse PCR (RT-qPCR) values for I-SceI expression.
Figure 1—figure supplement 1—source data 3. Red cells values after I-SceI induction.
Figure 1—figure supplement 1—source data 4. Agarose gel after PCR amplification of the I-SceI cutting site and cut by I-SceI in vitro.
Figure 1—figure supplement 1—source data 5. 2D distances values.