An FHA mutation suppresses Tel1 activity but not Mec1 activity during both mitosis and meiosis.

<p>A. Phosphorylation of Rad53 protein was analyzed at the indicated time points after phleomycin addition. Rad53 protein was detected by Western blotting by using anti-Rad53 in wild type (W303-1A), <i>xrs2-SH</i> (MSY2199), <i>rad50S</i> (MSY2203), <i>rad50S mec1</i>Δ (MSY2461) and <i>mec1</i>Δ <i>rad50S xrs2-SH</i> (MSY2372). Asterisks indicate phosphorylation signal of Rad53. Tubulin blots are shown as internal control for protein loading. B. Meiosis progression at the indicated time points after SPM transition in wild type (NKY1551), <i>rad50S</i> (MSY1758), <i>xrs2-SH</i> (MSY1867), <i>rad50S xrs2-SH</i> (MSY1844), <i>xrs2-664</i> (MSY2015), <i>rad50S xrs2-664</i> (MSY2106) and <i>rad50S tel1</i>Δ (MSY1952). The percentage of cells containing two or more nuclei per ascus (post-MI%) was plotted in the graphs. Images to the right show typical DAPI images after 6 hr of meiosis in each cell line, and the corresponding percentage of post-MI cells for each strain is shown. Scale bar indicates 2 μm. C. Meiosis progression in wild type (NKY1551), <i>xrs2-SH</i> (MSY1867), <i>dmc1</i>Δ (MSY2638) and <i>dmc1</i>Δ <i>xrs2-SH</i> (MSY4674). Typical DAPI micrographs after 6 hr of meiosis in each cell line, with addition of <i>mec1Δ dmc1</i>Δ as a control, are shown to the right. Scale bar indicates 2 μm. D. Phosphorylation of Hop1 at T318 (pT318) at the indicated time points after SPM transition was analyzed in wild type (NKY1551), <i>rad50S</i> (MSY1758), <i>xrs2-SH</i> (MSY1867), <i>rad50S xrs2-SH</i> (MSY1844), <i>xrs2-664</i> (MSY2015), <i>rad50S xrs2-664</i> (MSY2106) and <i>rad50S tel1</i>Δ (MSY1952). Hop1 protein was analyzed by western blot with anti-Hop1 (Hop1; green) or with anti-Hop1-pT318 antibody (pT318; magenta), and then each fluorescence signal was detected by laser scanner on the same membrane. Highly mobility shifted band was marked with asterisk. A merged image is shown on the top. Tubulin blots are shown as an internal control for protein loading. E. Localization of Hop1 (red) and Hop1-pT318 (green) on the meiotic nuclear spreads at 4 hr after SPM transition was analyzed in wild type (NKY1551), <i>rad50S</i> (MSY1758), <i>xrs2-SH</i> (MSY1867), <i>rad50S xrs2-SH</i> (MSY1844), <i>xrs2-664</i> (MSY2015), <i>rad50S xrs2-664</i> (MSY2106) and <i>rad50S tel1</i>Δ (MSY1952). Scale bar indicates 2 μm.