Fig. 7
From: Slx5-Slx8 ubiquitin ligase targets active pools of the Yen1 nuclease to limit crossover formation
Yen1-K714R increases COs and suppresses spontaneous chromosome segregation defects in mus81∆ cells. a Diagram explaining the Chr. XV DSB-induced crossover reporters. Recombination outcomes were scored in white/red sectored colonies of the indicated strains and normalized to its plating efficiency (PE). The number of independent experiment trials (T) and the total number of recombination events scored (n) were as follows: WT (T = 6, n = 158), yen1∆ (T = 9, n = 538), mus81∆ (T = 5, n = 168), mus81∆ yen1∆ (T = 9, n = 160), yen1-K714R (T = 5, n = 230), and mus81∆ yen1-K714R (T = 5, n = 230). b A strain harboring a lacO/GFP-LacI array tag on chromosome IV was followed by video-microscopy to discriminate chromosome segregation in timely manner from aberrant segregation. Images display a typical normal and aberrant segregation and its respective kymograph. c GFP foci of the indicated strains were observed by video-microscopy and chromosomal segregation was scored as to whether it displayed a proper phenotype (normal) or one of three types of defective phenotypes (non-disjunction, delay, and aberrant chromosome number (acn)). The total number of cells analysed (n) and independent video-recordings (VR) were as follows: WT (n = 606, VR = 3), yen1∆ (n = 133, VR = 3), yen1-K714R (n = 131, VR = 3), mus81∆ (n = 259, VR = 5), yen1-K714R mus81∆ (n = 448, VR = 3) and yen1∆ mus81∆ (n = 289, VR = 3). d Segregation events scored as in c were determined for mus81∆ and mus81∆ yen1∆ strains containing a pYES2 plasmid expressing Yen1-HA under Galactose-inducible control or an empty pYES2 and subjected to acute over-expression of Yen1-HA or the equivalent mock induction prior to the recording of the video-microscopy. The total number of cells analysed from three VRs were as follows: mus81∆ (+pYES2) n = 245, mus81∆ (+pYES2-Yen1) n = 391, mus81∆ yen1∆ (+pYES2) n = 117, mus81∆ yen1∆ (+pYES2-Yen1) n = 218. Statistically significant differences in a between CO and other outcomes and in c and d between normal and abnormal categories were determined by the Fischer’s exact test, asterisks refer to significance at the P < 0.001(***), P < 0.005(**) or P < 0.05(*)
