Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Japan.
PLoS One. 2012;7(1):e30472. doi: 10.1371/journal.pone.0030472. Epub 2012 Jan 26.
The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1(+) strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe.
组蛋白 H3-H4 伴侣蛋白 Asf1 参与染色质组装（或拆卸）、组蛋白交换、转录调控和几种生物的染色质沉默。为了研究 Asf1 在裂殖酵母中的必需功能，通过使用 PCR 的随机诱变构建了 asf1-ts 突变体。一个突变体（asf1-33(ts)）与 77 个不同激酶基因的突变体交配，以鉴定合成致死组合。asf1-33 突变体在限制温度下的存活需要 DNA 损伤检查点因子 Chk1 和 Rad3。在限制温度下，asf1-33 突变体中 Chk1 而不是 Cds1 被磷酸化，表明 DNA 损伤检查点在 asf1-33 突变体中被激活。asf1-33 突变体中发生了 DNA 损伤，通过脉冲场凝胶电泳观察到染色体 DNA 的降解，并形成 Rad22 焦点。与 asf1(+) 菌株相比，asf1-33 突变体在限制温度下对微球菌核酸酶的敏感性增加，表明 asf1 突变也导致整体染色质结构缺陷。与野生型相比，asf1-33 突变体蛋白定位错误且不能结合组蛋白。此外，asf1-33 突变体中着丝粒外重复区的组蛋白 H3 水平降低，异染色质结构受损。最后，鉴定出编码 CenH3 组蛋白伴侣蛋白的 sim3 为 asf1-33 突变体的强抑制子。总之，这些结果清楚地表明 Asf1 在维持裂殖酵母基因组稳定性方面发挥着重要作用。
