Fig. 7: The lack of Dpb3, but not of Isw2, impairs Rad9 association to DSB and checkpoint activation.

a DSB resection. YEPR exponentially growing cell cultures were transferred to YEPRG at time zero to induce HO production. SspI-digested genomic DNA was analyzed as in Fig. 2a. The experiment was performed independently three times with similar results; see Supplementary Figure 4 for the densitometric analysis of the resection products from three independent experiments as in a. b YEPR exponentially growing cell cultures of JKM139 derivative strains were transferred to YEPRG at time zero to induce HO. Protein extracts were subjected to western blot analysis with anti-Rad53 antibodies. c Phleomycin (10 μg/mL) was added to exponentially growing cells followed by western blot analysis with anti-Rad53 antibodies. The experiments in b and c were performed independently two times with similar results. d Exponentially growing YEPR cell cultures of JKM139 derivative strains were transferred to YEPRG to induce HO expression. Relative fold enrichment of Rad9-HA at the HO-induced DSB was evaluated after ChIP with anti-HA antibody and qPCR. The mean values of three independent experiments are represented with error bars denoting s.d. ***p < 0.005 (unpaired two-tailed Student’s t-test). e, f Serial dilutions of exponentially growing cultures onto YEPD plates with or without CPT. g HO expression was induced at time zero by galactose addition to G2-arrested cells that were kept arrested in G2 by nocodazole. Relative fold enrichment of Pol2-HA at the HO-induced DSB was evaluated after ChIP with anti-HA antibody and qPCR. The mean values of three independent experiments are represented with error bars denoting s.d. ***p < 0.005; **p < 0.01; *p < 0.05 (unpaired two-tailed Student’s t-test). h Western blot with anti-HA antibodies of protein extracts from G2-arrested cells. The experiment was performed independently three times with similar results.