Figure 2.

Histone H2A point mutants show chromosomal instability when released from mitotic arrest with nocodazole. (A) Nucleosome structure. Histones H2A, H2B, H3, H4, and DNA are yellow, red, blue, green, and white, respectively. (B, C) Enlarged views of the focused histone H2A C-terminal tail region (B) and the histone H3 residues located in proximity to H2A-I112 (C). Upper and lower panels represent the surface and cartoon models generated using the Pymol software application (see Materials and methods). (D) Multiple alignments of various histone H2A C-terminal sequences across species. Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Xl, Xenopus laevis; Mm, Mus musculus; Hs, Homo sapiens. Red circles indicate the histone residues conferring TBZ/benomyl sensitivity. (E) Histone H2A point mutants in the C-terminal region show perturbation of cell-cycle progression when released from mitotic arrest with nocodazole (Noc). Samples were taken at the times indicated and analysed by flow cytometry as described in Materials and methods. Asyn, asynchronous cells. (F) The spindle assembly checkpoint in H2A-I112A and -L117A cells is functional. A deletion mutant of Mad2, a spindle assembly checkpoint protein, was used as a positive control. Samples were harvested at the indicated times and analysed by flow cytometry as described in Materials and methods. α-Factor was used to arrest cells in the G1 phase. (G) Experimental scheme for figure (H). Cells were arrested in G1 phase by the addition of α-factor for 3 h, and released from G1 arrest in the presence of nocodazole. (H) According to the scheme shown in (G), cells were lysed at the indicated times after release from G1 arrest and analysed by immunoblotting for Pds1-3HA and histone H3 (control).