SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52
SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52
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DOI：
10.1093/nar/29.20.4179
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发表时间：
2001-10-15
影响因子：
14.9
通讯作者：
Watts, FZ
中科院分区：
文献类型：
--
作者：
Ho, JCY;Warr, NJ;Watts, FZ
粟酒裂殖酵母 rad31 和 hus5 基因是 DNA 损伤反应所必需的，因为这些基因缺陷的突变体对 DNA 损伤剂（例如紫外线和电离辐射）以及 DNA 合成抑制剂羟基脲 (HU) 敏感。序列分析表明，rad3l 和 hus5 编码粟酒裂殖酵母中 Pmt3 (SUMO) 修饰过程的成分。我们在此表明，rad3l null 和 hus5.62 突变体显示 Pmt3 修饰水平降低。我们已经开始寻找 DNA 损伤反应所需的蛋白质，这些蛋白质可能会被 Pmt3 修饰，并鉴定出 Rad22，即重组蛋白 Rad52 的裂殖酵母同源物。从表达 HA 标记的 Pmt3 的细胞中纯化 myc + His 标记的 Rad22 蛋白，鉴定出与抗 HA 抗血清交叉反应的 83 kDa 种类。我们在此表明 Rad22 与 Rhp51 和 Rpa70（分别是 Rad51 的裂殖酵母同源物和 RPA 大亚基）相互作用，但这些蛋白质似乎都不与 83 kDa 物种有关。当在天然和变性条件下制备提取物时，可以观察到 83 kDa 的种类，当 myc + His 标记的 Rad22 和 Pmt3 以野生型水平表达时，也可以观察到 83 kDa 的种类，表明 Rad22 在体内被 Pmt3 修饰。我们建立了裂殖酵母体外Pmt3修饰系统，并表明Rad22和Rhp51在体外被修饰，但Rpa70没有。
The Schizosaccharomyces pombe rad31 and hus5 genes are required for the DNA damage response, as mutants defective in these genes are sensitive to DNA damaging agents, such as UV and ionising radiation and to the DNA synthesis inhibitor hydroxyurea (HU). Sequence analysis has suggested that rad3l and hus5 encode components of the Pmt3 (SUMO) modification process in S.pombe. We show here that the rad3l null and hus5.62 mutants display reduced levels of Pmt3 modification. We have initiated a search for proteins required for the DNA damage response, which may be modified by Pmt3 and have identified Rad22, the fission yeast homologue of the recombination protein Rad52. Purification of myc + His-tagged Rad22 protein from cells expressing HA-tagged Pmt3 identifies an 83 kDa species which cross-reacts with anti-HA antisera. We show here that Rad22 interacts with Rhp51 and Rpa70 (the fission yeast homologues of Rad51 and the large subunit of RPA, respectively), but that neither of these proteins appears to be responsible for the 83 kDa species. The 83 kDa species is observed when extracts are prepared under both native and denaturing conditions, and is also observed when myc + His-tagged Rad22 and Pmt3 are expressed at wild type levels, suggesting that Rad22 is modified by Pmt3 in vivo. We have established an S.pombe in vitro Pmt3 modification system and have shown that Rad22 and Rhp51 are modified in vitro, but that Rpa70 is not.