Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 2

Figure 2. Cdc14 is required for inter-chromosomal DNA repair by HR but is not involved in DSB repair pathway choice.

A) Left panel: Schematic diagram displaying relevant genomic structure of the strains used to measure inter-chromosomal DNA repair efficiency. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that crossover and non-crossover product have different restriction fragment sizes that can be differentiated in a Southern blot assay. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 mutant strains carrying the inter-chromosomal gene conversion assay. Cells were grown overnight before adding galactose at semipermisive temperature to induce HO expression, 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 DNA sequence and an actin probe was used as loading control. Asterisk denotes an overexposed film to visualize crossover formation. Immunoblot of Rad53 was performed as previously described. Coomassie staining is shown as a control for loading. Graphs show the mean ± S.D. of gene conversion, DSB induction and crossover vs non-crossover ratio from three independent experiments. All data was normalized using actin as loading control. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.

B) Left panel: Schematic representation depicting the genomic structure of the strains used to evaluate repair pathway choice. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Note that the use of NHEJ or HR generates different product that can be recognized in Southern blots by the different disposition of the restriction sites for the endonucleases used. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of wild-type and cdc14-1 cells harbouring the repair pathway choice assay. Cells were grown overnight and HO induction was attained through addition of galactose for 1.5 hour at semipermisive temperature. After induction of the HO, glucose was added to repress the HO expression and samples were taken to analyse repair efficiency. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Blots were hybridized with a probe corresponding to the MATa-only DNA sequence. A second probe to the HIS3 gene was used to control the amount of genomic DNA loaded at each time-point. Graphs show quantification of mating type switching by gene conversion (HR), restoration of MATa (NHEJ) and the kinetics of the DSB induction. All data was normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.

Data information: Raf, raffinose. Gal, galactose. Glu, glucose. DSB, Double Strand Break. GC, gene conversion. CO, crossover. NHEJ, Non-homologous end joining.