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Dot1 is required for checkpoint-promoted localization and activation of Mek1.

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posted on 2013-01-31, 01:51 authored by David Ontoso, Isabel Acosta, Fred van Leeuwen, Raimundo Freire, Pedro A. San-Segundo

(A) Formation of zip1-induced Mek1 foci is defective in the absence of Dot1. Representative images of Ddc2-GFP and Mek1-GFP foci in zip1 and zip1 dot1 cells after 24 h in meiosis. Strains are DP460 (zip1 DDC2-GFP), DP579 (zip1 dot1 DDC2-GFP), DP582 (zip1 MEK1-GFP) and DP583 (zip1 dot1 MEK1-GFP). All strains are ndt80-arrested at pachytene. The graphs show the quantification of Ddc2 and Mek1 foci formation from the same samples determined as the intensity of the total focal GFP signal relative to total nuclear signal (a.u., arbitrary units). Error bars represent the median with interquartile range. Each spot in the plot represents the foci intensity of every nucleus measured. 175 and 150 nuclei were analyzed for Ddc2-GFP and Mek1-GFP, respectively. (B) Western blot analysis of Mek1 activation by phosphorylation and Cdc5 production throughout meiosis in wild type (DP421), zip1 (DP422), zip1 dot1 (DP555), zip1 dot1 GST-MEK1 (DP785) and zip1 GST-MEK1 (DP792) using Phos-tag gels. PGK was used as a loading control. (C) Analysis of Mek1 activation in ndt80-arrested cells. Strains are DP424 (wild type), DP428 (zip1) and DP655 (zip1 dot1). (D) Time course of meiotic nuclear divisions; the percentage of cells containing more than two nuclei is represented. Strains are: DP421 (wild type), DP422 (zip1), DP555 (zip1 dot1), DP785 (zip1 dot1 GST-MEK1), DP783 (zip1 dot1 GST-mek1-K199R), DP784 (zip1 dot1 GST-mek1-T327A), DP792 (zip1 GST-MEK1), DP790 (zip1 GST-mek1-K199R) and DP791 (zip1 GST-mek1-T327A).


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