DNA double-stranded breaks occurred in the <i>asf1-33</i> mutant.

<p>(A) Pulse-field gel electrophoresis analysis of the <i>asf1-33</i> mutant. L972 (<i>asf1⁺</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were incubated in YES medium at 26°C and 36°C for 6 h, and 5.0×10⁸ cells were collected by centrifugation. Pulse field gel electrophoresis was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. The intensity of DSB bands was measured using ImageJ. (B) Rad22-GFP foci in SKP558-7 (<i>asf1⁻</i>) and KT166 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were observed after incubation at 26°C or 36°C for 6 h. Fluorescence of Rad22-GFP foci was observed with a Leica TCS-SP5 confocal laser scanning microscope (Leica Microsystems, Japan). Arrows indicate the Rad22-GFP foci. (C) The number of Rad22-GFP foci was counted.