Fig. 8: Cdc14 facilitates DNA re-synthesis and extension of GC tracts.

a Coverage levels at 0.5 kb (left panels) and 15 kb (right panels) at both sides of the HO cleavage site in wild-type (blue) and cdc14-1 (red) PMV cells from 4 h after the endonuclease expression. Synthesis rates (S) between different time intervals are indicated (see Supp. Figure 6b for details). Light blue/red: left HO side. Dark blue/red: right HO side. The graphs show the mean ± SD from two biologically independent experiments. b Relative proportion of reads containing polymorphisms in the MATa and MATa′ regions of chromosomes III and V, before (T0) and after (T10/T24) induction of the HO endonuclease in wild-type (blue) and cdc14-1 cells (red). Horizontal red lines mark the expected profile of polymorphisms in MATa and MATa′ considering the total DNA repair levels obtained by 10 h (wild-type) and 24 h (cdc14-1). The graphs show the mean ± SD from two biologically independent experiments. c Graphs representing the coordinates between MATa and MATa′ discordant read pairs in wild-type (top) and cdc14-1 cells (bottom). Intersection of MATa/MATa′ coordinates on each quadrant represent distinctive GC boundaries (see diagrams). Discordant reads pairs falling at the left-bottom quadrant and right-bottom quadrant correspond, respectively, to the left and right boundaries of GC events taking place at the left side of the HO break. Discordant reads pairs falling at the left-top quadrant and right-top quadrant correspond, respectively, to the left and right boundaries of GC events taking place at the right side of the HO break. The top blue and right red coverage profiles are generated by filtering MATa and MATa′ reads from the discordant pairs and aligning them to the reference genome. d Overlap of the MATa and MATa′ coverage profiles obtained from the analysis shown in c in wild-type (top panel) and cdc14-1 (bottom panel) cells. Blue and red represent, respectively, the read coverage profile of MATa and MATa′ reads isolated from the MATa/MATa′ discordant read pairs identified. A, B, C, D, and E represent the relative position of the discordant read pairs along the MAT locus after repair. e Blue and red densitometry panels displaying coverage levels of both MATa and MATa′ loci, respectively. Graph represents the percentage of A-E discordant read pairs relative to the total number of events. Graph shows the mean ± SD from two biologically independent experiments. P-values were calculated using a two-tailed unpaired Student′s t-test. *: P ≤ 0.05, **: P ≤ 0.01; ***: P ≤ 0.001. Black arrows mark the position of the HO cleavage sites. f Measurement of the maximum extension of GC at both sides of the HO break in wild-type and cdc14-1 PMV cells. The coverage profiles shown in d were used to determine the boundaries of GC generated during the incorporation of MATa′ polymorphisms into the MATa locus in wild-type (blue) and cdc14-1 (red) cells. A, B, C, D, and E represent the relative position from the HO site (nucleotides) of the discordant read pairs along the MAT locus after repair. Black arrow marks the position of the HO cleavage sites.