Figure 5. The lack of γH2A enhances transcriptional inhibition around the DSB by accelerating resection.

(A) YEPR exponentially growing cell cultures of the JKM139 derivative strains, carrying the HO cut site at the MAT locus, were transferred to YEPRG at T0. RNA levels of genes located in the surroundings of the HO cut site at the MAT locus were analyzed at T0 and T240 after HO induction by qRT-PCR as described in Figure 2A. The mean values ±s.d. are represented (n = 3). (B) Method to measure DSB resection. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded MAT probe (ss probe) that anneals to the unresected strand. 5′–3′ resection progressively eliminates SspI sites (S), producing larger SspI fragments (r1 through r7) detected by the probe. (C) DSB resection. Genomic DNA prepared from samples collected in (A) was analysed for single-stranded DNA (ssDNA) formation at the indicated times after HO induction as described in (B). The image that was used for the cropped final Figure 5C is available in Figure 5—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.08942.008