Fig. 5
From: Slx5-Slx8 ubiquitin ligase targets active pools of the Yen1 nuclease to limit crossover formation
Yen1 foci are dynamic and localize preferentially to nucleolar sites in the absence of DNA damage. a slx8∆ cells carrying a SIK1-mCherry endogenous marker and an inducible GFP-Yen1 expressing plasmid were observed after short induction of the fusion protein. The white arrow denotes co-localizing signal of GFP-Yen1 with Sik1-mCherry. b Wild-type cells carrying a TetO-TetR array tag on chromosome XII and an inducible GFP-Yen1 expressing plasmid were observed after short induction of the fusion protein. c Cells were subjected to acute challenge with Zeocin (0.01 mg/ml) and observed during their recovery as in Fig. 4. Cells displaying the designated categories of GFP-Yen1 foci were scored at the indicated time points. The total number of cells analysed (n) from two independent recordings were as follows: WT 0 h (nG1 = 81, nG2/M = 267), WT 1.5 h (nG1 = 78, nG2/M = 124), WT 3.5 h (nG1 = 110, nG2/M = 118), slx8∆ 0 h (nG1 = 107, nG2/M = 79), slx8∆ 1.5 h (nG1 = 40, nG2/M = 73), slx8∆ 3.5 h (nG1 = 52, nG2/M = 62). d Cells were subjected to an acute challenge with 0.1% MMS and for foci were observed as in c. The total number of cells analysed (n) from two independent recordings were as follows: WT 0 h (nG1 = 81, nG2/M = 267), WT 1.5 h (nG1 = 95, nG2/M = 98), WT 3.5 h (nG1 = 139, nG2/M = 137), slx8∆ 0 h (nG1 = 107, nG2/M = 79), slx8∆ 1.5 h (nG1 = 53, nG2/M = 55), slx8∆ 3.5 h (nG1 = 40, nG2/M = 67). e slx8∆ cells carrying SIK1-mCherry were observed after Zeocin challenge to determine GFP-Yen1 co-localization. White arrows indicate GFP-Yen1 foci. f slx8∆ cells were observed as in e, but were subjected to MMS treatment. White arrows indicate GFP-Yen1 foci. Images are representative of the reproducible results obtained after three independent trials. Statistical significance at P < 0.0001 in Fischer’s exact test at 3.5 h recovery points is indicated by asterisks in c and d
