Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama, Japan.
FEBS J. 2010 May;277(10):2360-74. doi: 10.1111/j.1742-4658.2010.07651.x.
Meiotic recombination is initiated by DNA double-stranded breaks introduced by the SPO11 protein. Despite a decade of research, the biochemical functions of SPO11 remain largely unknown, perhaps because of difficulties in studying the functionally active SPO11. Arabidopsis thaliana encodes three SPO11-related proteins, two of which (SPO11-1 and SPO11-2) are required for, and cooperate in, meiosis. We isolated soluble SPO11-1, fused with or free of a trigger factor-tag at its N terminus. The tag-free SPO11-1 needed to interact physically with soluble SPO11-1 to maintain its solubility, suggesting a multimeric active form including a solubilizing protein cofactor. An N-terminal fragment of PRD1, a SPO11-1-interacting protein required for normal meiosis, but not SPO11-2, forms a soluble complex with trigger factor-tagged SPO11-1, but the trigger factor-tag was required for the solubility. Formation of the complex is not sufficient to express endonuclease activity. Trigger factor-tagged SPO11-1 exhibited DNA-binding activities: Glu substitutions of the invariant Gly215 and Arg222 and of the nonconserved Arg223 and Arg226 in a conserved motif (G215E, R222E, R223E, R226E) reduced the DNA-binding ability in vitro, but substitutions of the conserved Arg130 and invariant Tyr103 (a residue in the putative endonuclease-active center) and of Arg residues outside conserved motifs by Glu or Phe (R130E, Y103F, R207E and R254E), did not. Tests for the ability of mutant spo11-1 proteins to complement the silique-defective phenotype of a spo11-1-homozygous mutant in vivo revealed that R222E and G215E induced serious deficiencies, while R130E caused a partial defect in silique formation. Thus, the Gly215, Arg222 and Arg223 residues of SPO11-1 form a DNA-binding surface that is functional in meiosis.
减数分裂重组是由 SPO11 蛋白诱导的 DNA 双链断裂启动的。尽管经过了十年的研究，SPO11 的生化功能在很大程度上仍然未知，这也许是因为研究具有功能活性的 SPO11 存在困难。拟南芥编码三种 SPO11 相关蛋白，其中两种（SPO11-1 和 SPO11-2）对于减数分裂是必需的，并在减数分裂中合作。我们分离了可溶性 SPO11-1，其 N 端融合或不融合触发因子标签。无标签的 SPO11-1 需要与可溶性 SPO11-1 物理相互作用才能保持其可溶性，这表明存在包括可溶性辅助因子在内的多聚体活性形式。PRD1 的 N 端片段是 SPO11-1 相互作用蛋白，对于正常减数分裂是必需的，但不是 SPO11-2，与带触发因子标签的 SPO11-1 形成可溶性复合物，但该标签对于可溶性是必需的。复合物的形成不足以表达内切核酸酶活性。带触发因子标签的 SPO11-1 表现出 DNA 结合活性：在保守基序（G215E、R222E、R223E、R226E）中，不变的 Gly215 和 Arg222 以及非保守的 Arg223 和 Arg226 的 Glu 取代降低了体外 DNA 结合能力，但保守的 Arg130 和不变的 Tyr103（假定内切核酸酶活性中心的残基）以及 Arg 残基的 Glu 或 Phe 取代（R130E、Y103F、R207E 和 R254E）并没有。体内测试突变 spo11-1 蛋白弥补 spo11-1 纯合突变体在 siligue 缺陷表型的能力的实验表明，R222E 和 G215E 引起严重缺陷，而 R130E 导致 siligue 形成的部分缺陷。因此，SPO11-1 的 Gly215、Arg222 和 Arg223 残基形成了在减数分裂中起作用的 DNA 结合表面。
