Fig. 1: Cdc14 phosphatase is required for recombinational DNA repair.

a Schematic representation showing the relevant genomic structure of the PMV strain. The HMLα and HMRa loci at chromosome III have been deleted to drive MATa recombination to a 1.3 kb MATa′ fragment located at the ARG5,6 locus in chromosome V that contains 23 polymorphisms (purple lines). Three of the MATa′ polymorphisms generate EcoRI sites (ERI) at both sides of the uncleavable HO-inc site (blue lines), facilitating the analysis of GC by Southern blot experiments. The size of the “Uncut” and “Cut” DNA fragments generated after inducing the HO endonuclease are shown. The probes used to determine the incorporation of the EcoRI sites from chromosome V into III by Southern blot are shown. b Diagram representing the hypothetical GC outcomes that could be generated during the repair of the HO-induced break. Horizontal red lines represent the position of the left and right probes. Vertical black lines represent the EcoRI sites on chromosome III. Vertical blue lines denote the potential EcoRI sites incorporated from the MATa′ into the MATa locus after GC. The size of the bands generated when digesting with EcoRI for each possible GC outcome is shown. c Southern blot analysis of DNA repair in wild-type and cdc14-1 PMV cells. The strains were grown in YP-Raffinose at 25 °C and alpha factor (αF) was added to block the cells in G1. After the arrest, cells were liberated and galactose was added to the media to induce HO expression. One hour after HO induction, cultures were transferred to 33 °C and samples were taken at the indicated time points. DNA was extracted, digested with EcoRI and analyzed by Southern blot using the left (top panel) and right (bottom panel) probes shown in a. The DNA molecular weights on the left correspond to the distinctive DNA fragments shown in a. An ACT1 gene sequence was used as a loading control. RI: resection intermediates. Diagrams on the right represent EcoRI sites (blue dots) surrounding the HO cleavage site (red bar) transferred from the MATa′ to the MATa locus as depicted in b. The graphs on the right represent the quantification of the different GC outcomes obtained after repair. The mean ± SD from three independent Southern blots experiments after normalization against their respective ACT1 signal and uncut T0 sample is shown.