Skip to main content
. 2016 Nov 16;36(1):79–101. doi: 10.15252/embj.201593540

Figure EV5. Low Spc110 activity results in high SPB dynamics, loss of DSB‐SPB interaction and inefficient DSB repair by homologous recombination.

Figure EV5

  1. D1, D2 and D3 distances measured in wild‐type and spc110‐220 cells after inducing a non‐reparable DSB at the MAT locus. Cnm67‐CFP and Ddc2‐RFP were used as SPB and DSB markers, respectively. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z‐planes. P‐values were calculated using a two‐tailed unpaired Student's t‐test. A diagram with the genome information and a representative picture are shown. Scale bar: 3 μm.
  2. Time‐lapse experiments in living cells were carried out after expressing the HO endonuclease in wild‐type and spc110‐220 cells. Three z‐planes every 10‐s intervals over a period of 5 min were captured and used to quantify the average SPB track length and velocity using Spc110‐RFP as SPB marker. At least 100 cells were scored.
  3. Physical analysis of wild‐type and spc110‐220 mutant strains carrying the inter‐chromosomal gene conversion assay depicted (top diagram). Cells were grown overnight and exposed to HO expression by adding galactose at 32°C, thus producing a DSB on chromosome V. Samples were taken at different time points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were hybridized with a MATa‐only and ACT1 (loading control) DNA sequences. FACS profiles for DNA content are included. Graphs represent quantification of gene conversion, DSB induction and crossover versus non‐crossover ratio. All data were normalized with the actin control. Graphs show the mean ± SD from three independent experiments. P‐values were calculated using a two‐tailed unpaired Student's t‐test. Asterisk denotes an overexposed film to visualize crossover formation. Data information: DSB, double‐strand break; SPB, spindle pole body; Raf, raffinose; Gal, galactose; GC, gene conversion; CO, crossover.

Source data are available online for this figure.