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. 2013 Jul 30;41(19):8943–8958. doi: 10.1093/nar/gkt645

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Mus81-Mms4 regulation is not modified in cells lacking Sgs1 or Yen1. (A) sgs1ΔHA-MMS4 cells (YMG21 strain) were synchronized in G1 and then released into fresh medium ±MMS (0.02%). Cell cycle progression was monitored by flow cytometry (left). Immunoblot analysis of Mms4 (right). (B) G1-blocked sgs1Δyen1ΔHA-MMS4 cells (YSG56 strain) were released into fresh medium ±MMS (0.02%). Left: Flow cytometry. Right: Immunoblot analysis of Mms4. (C) Cell cycle experiments for the nuclease assays. Cells were synchronized in G1 and released in medium with MMS (0.02%) for 60 min or in medium without MMS (plus nocodazole). (D) The extracts were prepared from cells taken at the indicated time points and HA-Mms4 was immunoaffinity purified. Mms4 was analysed by immunoblot in the whole cell extracts (WCE), and the yield of the IP was estimated. About 2% of the total amount of the IP protein used for the nuclease assays was loaded. (E) The nuclease activity was assayed by the resolution of a model RF and a 3′-FL. The arrows indicate the labelled products resulting from the cleavage of each substrate. The controls were a reaction without extract and a nuclease assay using IP-extracts from untagged cells blocked in G2/M.