Fig. 2: Lack of Cdc14 produces over-resection of DSBs.

a Schematic representation of the DNA resection assay used to determine the extent of resection in the PMV background. The size of the bands for the “Uncut”, “Cut”, “Donor”, and the different distances analyzed from the HO cleavage site are shown. The probes used to determine the kinetics of these bands through the repair of the HO-induced break are depicted with horizontal red lines. S: StyI. b Southern blot analysis of wild-type and cdc14-1 PMV cells using the experimental approach shown in a. After overnight culture in YP-Raffinose at 25 °C, alpha factor (αF) was added to block the cells in G1. After arrest, cells were released by removing the pheromone, induced with galactose, and transferred to 33 °C. Samples were taken at different intervals after HO induction, and genomic DNA was extracted, digested with StyI and analyzed by Southern blot using the probes depicted in a. A MET5 gene sequence was used as a loading control. The graphs on the right represent the quantification of the averaged band signals from two Southern blots after normalization against their respective MET5 signal and uncut T0 sample. Note that in both strains, proximal probes retrieved to a higher level before that of the distal probes from the HO-induced break, a feature that might account for DNA repair by SDSA. U/R: Uncut/Repair. c Schematic representation depicting relevant genomic structures of the YMV80 background used to assess DNA repair by SSA/BIR. The location of the HO cleavage site at the LEU2 locus (LEU2::cs) and the donor sequence (U2) are depicted. The size of the bands for the “Donor”, “Uncut”, “Cut”, “Repair” and the different distances used to assess resection dynamics (27 kb and 42 kb to the right of the HO site) are shown. The location of the probes (horizontal red lines) and the restriction endonuclease cleavage sites (K: KpnI) used for Southern blot analysis are illustrated. d Southern blot analysis of wild-type and cdc14-1 YMV80 cells using the experimental approach shown in c. After overnight culture in YP-Raffinose at 25 °C, cells were induced with galactose and transferred to 33 °C. Samples were taken at different intervals after HO induction, genomic DNA was extracted, digested with KpnI and analyzed by Southern blot using the probes depicted in c. A IMA5 gene sequence was used as a loading control. The graphs on the right represent the quantification of the averaged band signals from two Southern blots after normalization against their respective IMA5 signal and uncut T0 sample. Quantification of the product formation was attained by normalizing against the IMA5 and the T24 sample of the wild-type strain.