Figure 5.

KEOPS complex is recruited to the chromatin at DNA breaks. (A) Chromatin recruitment of Bud32 after MMS treatment. The chromatin was extracted from cells exposed to 0.05% MMS for indicated time points. The whole-cell extract (WCE) and chromatin-bound proteins were analyzed by western blot. (B) Schematic of the HO mediated DSB in the ‘donorless’ strain and real-time PCR primer sets on chromosome III. (C) The absolute fold enrichment of Bud32–13Myc and Kae1–13Myc at indicated distances from the HO recognition site in the absence of a DSB. –Ab and +Ab mean ChIP assay without and with anti-Myc antibody, respectively. Data are means ± s.e.m. (n = 3). (D, E) Relative fold enrichment of Bud32–13Myc (D) and Kae1–13Myc (E) at the indicated distances from the HO cleavage site after HO induction for indicated times. ChIP assays without antibody (–Ab) or with anti-Myc antibody (+Ab) were performed and real-time PCR was used to measure the enrichment. Data are means ± s.e.m. (n = 3). * represents P < 0.05, ** represents P < 0.01 (Student's t-test). (F) SDS-PAGE and Commassie blue staining analysis of purified yeast KEOPS complex. (G) DNA binding ability analysis by electrophoretic mobility shift assay. 2 nM of radio-labeled dsDNA or ssDNA was incubated with purified yeast KEOPS proteins (0, 0.4, 0.7, 1.0, 1.4 and 1.8 μM).