FIG 1.

Genotoxic stress inhibits G₂/M-specific gene transcription by preventing Ndd1 promoter recruitment. (A) Induction of genotoxic stress by HU or MMS downregulates G₂/M-specific gene transcription. An NDD1-HA strain (MK155) was synchronized in G₁ using α-factor and released into YEPD alone and YEPD with 0.1 M HU or 0.0075% MMS. CLB2 transcription was analyzed using Northern blot analysis from samples isolated at the indicated time points. Error bars indicate standard deviations in three independent experiments. fold to, fold expression of CLB2 relative to that of CMD1. (B) DNA content of cells from the experiment whose results are presented in panel A analyzed by FACS. (C) Recruitment of Ndd1 to G₂/M promoters is prevented by genotoxic stress. Ndd1 recruitment to the CLB2 promoter was examined by ChIP, followed by qPCR analysis from samples for which the results are described in panel A. Error bars indicate standard deviations in three independent experiments. (D and E) The Ndd1 protein is abundant and localized to the nucleus in HU- and MMS-treated cells. (D) MK155 cells synchronized in G₁ were released into YEPD with or without HU or MMS. The Ndd1-HA levels in samples isolated at the indicated time points were detected by Western blotting. Western blotting for alpha-tubulin was performed as a loading control. (E) A wild-type strain expressing NDD1-GFP from the GAL1-10 promoter (SKY293) was synchronized in G₁ and released into YEP with or without HU or MMS. NDD1-GFP expression was induced by galactose addition 30 min prior to release. Cells were fixed at 60 min postrelease, and nuclei were stained with Hoechst. Ndd1-green fluorescent protein (GFP) localization was examined by fluorescence microscopy. DIC, differential inference contrast.