FIG. 7.

DNA replication arrest in S phase with HU activates p53 expression and inhibits Hbo1 HAT activity. (A) An asynchronously dividing culture was split and either left untreated or arrested with 1.5 mM HU treatment. Cells were harvested from the cultures after 6 h of HU treatment and assayed by flow cytometry. The DNA content histograms are plotted as shown. (B) MCF7 cells were either left untreated (lane 1) (“−”) or treated for 6 h with 1.5 mM HU (lane 2) (“+”) as described above (A). Whole-cell extracts were separated by PAGE and assayed by Western blotting with antibodies against p53, Hbo1, p21, and α-tubulin as a loading control. The pairs of Western blot bands presented in columns 1 and 2 are from nonadjacent lanes of the same gel images. (C) MCF7 whole-cell extracts as described above (A) were immunoprecipitated with anti-Hbo1 antibody. Portions of the Hbo1 immunoprecipitates were then assayed by Western blotting for the presence of Hbo1 and p53 (“p53/Hbo1 in the complex”) and for HAT activity by [³H]acetate incorporation (“Fluorography”) into chicken core histones (“Stain”). (D) MCF7 cells, either untreated or HU arrested as described above (A), were fractionated into total cell extract (TCE) (lanes 1 and 2), a soluble cytoplasmic fraction (S2) (lanes 3 and 4), a soluble nuclear fraction (S3) (lanes 5 and 6), and a chromatin-enriched fraction (P3) (lanes 7 and 8) (43). Fractions were separated by PAGE and assayed by Western blotting with antibodies against p53, Mcm2, Mcm6, Hbo1, geminin, Cdt1, Orc2, and Mek2.