Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE
Fig 4
The nse3-1 allele exhibits genetic interactions with the loss of SIR4 and RIF2.
(A) TPE was determined in strains with the URA3 reporter at the adh4 locus of Chromosome VIIL as in Fig 3E. Overnight cultures were spotted onto SC (complete medium) and SC + .1% 5-FOA plates at 34°C in wild type (JC1991), sir4Δ (JC3818), nse3-1(JC3860), nse3-1 sir4Δ (JC3870) isogenic strains. (B) Transcription levels in wild type (JC470), nse3-1 (JC3607), sir4Δ (JC3737), and nse3-1 sir4Δ (JC3741), and (C) rif2Δ (JC2992) and nse3-1 rif2Δ (JC3269) at sub-telomeric genes CHA1 and VAC17 on Tel3L and YR043C on Tel9R as described in [62, 63]. Expression values are mRNA levels relative to ACT1 and normalization to wild type cells. Error bars represent ± SD of n = 3 experiments with p values < .05 from a two-tailed t-test indicated by (*). (D) Chromatin immunoprecipitation (ChIP) was performed on Rif2Myc and showed similar levels of recovery in wild type (JC2380) and nse3-1 (JC3235) mutants. (E) ChIP on Smc6FLAG in wild type (JC1594), rif1Δ (JC2754) and rif2Δ (JC3074) cells with enrichment levels for untagged strains in wild type and mutants shown in S5C and S5D Fig. The mean ± SD of the fold enrichment at three native subtelomeres (Tel1L, Tel6R and Tel15L) relative to the control (ctrl) late replicating region on Chromosome V (469104–469177) is reported. In rif2Δ mutants the p values < .05 = 0.53 (Tel1L), 0.13 (Tel6R), and 0.15 (Tel15L) indicated that the difference was not significant from wild type. (F) Telomere length was determined as previously described [15]. Southern blot analysis was performed on 1μg XhoI-digested genomic DNA hybridized with a radiolabeled poly (GT/CA) probe in wild type (JC470), nse3-1 (JC3607), rif1Δ (JC3448), nse3-1 rif1Δ (JC3623), rif2Δ (JC2992), nse3-1 rif2Δ (JC3269), rad52Δ (JC1427), nse3-1 rad52Δ (JC3629), rif2Δ rad52Δ (JC3603), and nse3-1 rad52Δ rif2Δ (JC3627) strains.