Fig. 1.

The PCNA-unloading activity of Elg1 contributed to silencing HML. (A) Illustration of the CRASH assay to measure silencing of HML. The CRASH assay contains two features: (i) Cre inserted at the HML locus controlled by the α2 promoter and (ii) an RFP-GFP reporter cassette on chromosome V at the URA3 locus. Cells that maintain silencing of HML constitutively express RFP driven by the GPD promoter. Loss of silencing at HML leads to Cre expression and results in Cre-mediated recombination between two LoxP sites, resulting in removal of the RFP and HygMX genes, repositioning the GFP gene so that it becomes constitutively expressed. (B) Representative images of colonies from wild-type (JRY10790) or elg1Δ mutant (JRY10799) strains containing the CRASH assay. (C) The apparent silencing-loss rates of the strains in B were quantified by flow cytometry, as described in Materials and Methods. A P value was calculated using an unpaired, two-sided t test. (D) Representative images of colonies from wild type (JRY10790) and pol30-D150E (JRY10800), elg1Δ (JRY10799), and elg1Δ pol30-D150E (JRY10801) mutant strains containing the CRASH assay. (E) Quantification of apparent silencing-loss rates by flow cytometry of the strains in D. P values were calculated by ANOVA and Tukey’s test post-hoc analysis. In C and E, the red line in the box plots represents the median value calculated from at least 10 cultures. The blue boxes represent the 25th and 75th percentiles. Whiskers represent the range of values within 1.5× the interquartile range. Values extending beyond 1.5× the interquartile range are marked as outliers (red plus sign).