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. 2014 Oct 9;42(20):12469–12482. doi: 10.1093/nar/gku927

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — Top2 and condensin are required for SAC activation independently of DNA damage in cells partially depleted of histones. (A) Top2 is required for SAC activation by histone depletion. Cell cycle progression was analyzed by flow cytometry of wild-type, M::HHF2, top2^td and M::HHF2 top2^td cells synchronized in G1 under restrictive conditions to deplete Top2 and then released into fresh medium under conditions of histone depletion. (B) Western blot analysis of Top2 and Ubr1 in top2^td and M::HHF2 top2^td G1 synchronized cells under restrictive conditions for Top2 and H4. Both proteins were labeled with Myc epitopes. Pgk1 was used as loading control. MW, molecular weight marker. (C) Condensin is required for SAC activation by histone depletion. Cell cycle progression was analyzed by flow cytometry of wild-type, t::HHF2, smc2-8 and t::HHF2 smc2-8 cells synchronized in G1 at permissive temperature (26°C) and released into fresh medium at restrictive temperature (37°C) under conditions of histone depletion. (D) Western blot analysis of Brn1 in wild-type and G::HHF2 cells synchronized in G1 and released into fresh medium under conditions of histone depletion in the presence of 15 μg/ml NCD for 90 min. Pgk1 was used as loading control. (E) A reduction in condensin activity partially suppresses t::HHF2 growth defects at 26°C. (F) Cells partially depleted of histone H4 are sensitive to Top2 overexpression, as determined by cell growth for wild-type and t::HHF2 cells transformed with plasmids YCp50 (empty) or YCpPDED1T2 (Top2). (G) Rad52 foci accumulation by histone depletion is independent of Top2. Rad52-YFP foci accumulation was determined in wild-type, M::HHF2, top2^td and M::HHF2 top2^td cells transformed with p314R52YFP (RAD52-YFP), synchronized in G1 under restrictive conditions to deplete Top2 and released into fresh medium for 60 min under conditions of histone depletion. (H) Rad52 foci accumulation in asynchronous cultures of wild-type, t::HHF2, smc2-8 and t::HHF2 smc2-8 cells transformed with pWJ1344 (RAD52-YFP) and grown at permissive temperature (26°C) under conditions of histone depletion (0.25 μg/ml). (G, H) A total number of 100 cells were analyzed for each strain and experiment. The average and SEM of three independent experiments performed with three different transformants from two independent transformation of each strain are shown. Asterisks indicate statistically significant differences (M::HHF2 and M::HHF2 top2^td relative to wild type and top2^td, and t::HHF2, smc2-8 and t::HHF2 smc2-8 relative to wild type) according to a one-way ANOVA test, where two and three asterisks indicate P < 0.01 and <0.001, respectively.