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

Figure 7. Microtubule destabilization affects DSB‐SPB interaction and DSB repair by homologous recombination.

Figure 7

  1. Cells treated with nocodazole are affected in DSB‐SPB interaction. Cnm67‐CFP and Ddc2‐RFP were used as SPB and DSB markers, respectively. Diagram depicts the genomic background used. D1: inter‐SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulphoxide (DMSO) were scored and plotted. 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 representative picture is shown. Scale bar: 3 μm.
  2. Physical analysis of wild‐type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α‐factor pheromone and released into fresh media for 1 h. Induction of HO expression was attained by adding galactose for 2 h. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa‐distal sequence and the HIS3 gene (as a loading control) were used. FACS analyses are shown. Graphs show the quantification of MATa restoration (NHEJ), mating‐type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± SD from three independent experiments. P‐values were calculated using a two‐tailed unpaired Student's t‐test.
Data information: DSB, double‐strand break; SPB, spindle pole body; DMSO, dimethyl sulphoxide; Nz, nocodazole; GC, gene conversion; Raf, raffinose; Gal, galactose; Glu, glucose.Source data are available online for this figure.