Fig. 4
From: Slx5-Slx8 ubiquitin ligase targets active pools of the Yen1 nuclease to limit crossover formation
Deletion of SLX8 alters the nuclear distribution and turnover of a fraction of Yen1. a Wild type and slx8∆ cells were synchronized in G1 and released to observe the phosphorylation of Yen1 as a function of cell-cycle progression. b Serial dilutions of the indicated strains were spotted onto YPAD media containing different genotoxins. c Cells with an endogenous HTA1-mCherry carrying plasmids expressing wild-type GFP-Yen1 were observed microscopically after a short induction of the fusion protein. Shown are cells presenting normal nuclear localization (lower) or presenting foci (white arrows, upper). d G1 and G2/M cells of the indicated genetic backgrounds were microscopically examined as in c and classified according to the number of foci they displayed. The graphs show the percentage of cells in each category. The total number of cells individually scored from three video recordings are indicated as (n). Categories were subjected to the Fischer’s exact test, asterisks denote significant levels at P < 0.001(***) or P < 0.005(**). e The duration of foci in the indicated genetic backgrounds was measured by video-microscopy analysis. The mean ± s.d. of the duration time and n are indicated, error bars denote s.d. Asterisks refer to significance at the P < 0.05 (**) and P < 0.001 (***) levels in unpaired two-tailed Student’s t-test. f Cells expressing GFP-Yen1 and Hta1-mCherry were observed by video-microscopy in 2′ time-lapse frames. GFP total intensity of the whole cell, the nucleus and the cytoplasm was determined for 5 z-planes and used to calculate the total GFP intensity in each compartment. The graph displays the time course of GFP intensity in a single cell g The indicated pdr5∆ strains, that are permeable to MG132, were synchronized in G1 and subjected to cycloheximide (CHX) treatment during their release from G1 arrest. Where indicated, cells were pre-treated with MG132 for 30 min before, and during release in the presence of CHX. PGK1 was used to normalize the amount of Yen1. h Quantitation of the fraction of Yen1, compared to G1, remaining at the indicated times after release into CHX. The mean ± s.d. of triplicate assays is shown; statistically significant difference in unpaired two-tailed Student’s t-test is indicated (**P < 0.05). i FACS analysis of cells at the beginning and at the end of the CHX treatment
