Figure 4.

Yku overproduction impairs 5′-end processing and Mre11 loading at an HO-induced double-strand break. (A–E) YEP+raf nocodazole-arrested cell cultures of JKM139 strains carrying either empty vectors or both the 2μ URA3 YKU70 and 2μ LEU2 YKU80 plasmids, all expressing the MRE11-MYC allele, were transferred to nocodazole-containing YEP+raf+gal (time zero). (A) DSB resection analysis as described in Fig 1B. (B) Densitometric analysis of the representative experiment shown in (A). Three independent experiments were performed with similar results. (C) Representative ChIP time-course analysis performed as described in Fig 2E. (D) Quantitative analysis of Mre11–DSB association. Densitometric data from three independent experiments as in (C) were expressed as in Fig 2F. Error bars indicate s.d. (E) Western blot analysis of protein extracts with Rad53 antibodies. (F) Exponentially growing YEP+raf cell cultures of mec1Δ LSY1259 (10 Ty1-HOcs-HIS3) strains transformed with 2μ URA3 plasmids, either empty or carrying both YKU70 and YKU80, were transferred to YEP+raf+gal to induce HO expression (time zero). Western blot analysis of protein extracts with Rad53 antibodies is shown. ChIP, chromatin immunoprecipitation; CON, control; DSB, double-strand break; Exp, exponentially growing cells; YEP, yeast extract peptone.