FIG. 2.

The pH2AX-binding motif of Crb2 is required for accumulation at IR-induced DSBs and cell survival after DNA damage. (A) Strains harboring a GFP-tagged crb2 allele were processed for live cell imaging either immediately before (0 Gy), immediately after (36 Gy), or 1.5 h after (1.5 h Post 36 Gy) IR exposure. Left shows a panel of representative images from cells either untreated (0 Gy) or immediately after irradiation (36 Gy). Right, quantification of nuclei with one or more Crb2 foci. Data are averaged from results of three independent experiments in which 200 to 300 cells were counted for each point. (B and C) Crb2 Lys619 mutations induce hypersensitivity to DNA damage. (B) Strains harboring the indicated mutation at the endogenous crb2 allele were grown in liquid culture, and fivefold serial dilutions were spotted onto either rich medium or rich medium containing the indicated amounts of CPT or HU. Cells spotted onto rich medium were then untreated (control) or irradiated with the indicated dose of IR (in grays [Gy]) or UV (in joules [J]/m²). All plates were then incubated at 30°C. For panel C, quantitative IR survival curves were performed. Log-phase cells grown in liquid culture at 30°C were treated with the indicated amount of IR (x axis), and 600 to 800 cells were plated in duplicate onto rich medium plates. Colony formation was measured after incubation at 30°C, and survival was plotted relative to the unirradiated 0 Gy sample (y axis). Data are averaged from results of three independent experiments with a standard error of 10% or less. (D) Expression of mutant Crb2 proteins. Total cell lysates were prepared from the top-labeled strains and processed for Western blotting using the indicated (left) polyclonal antibodies.