Matsubara Kazuko, Sano Norihiko, Umehara Takashi, Horikoshi Masami
Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Genes Cells. 2007 Jan;12(1):13-33. doi: 10.1111/j.1365-2443.2007.01031.x.
The core histones are essential components of the nucleosome that act as global negative regulators of DNA-mediated reactions including transcription, DNA replication and DNA repair. Modified residues in the N-terminal tails are well characterized in transcription, but not in DNA replication and DNA repair. In addition, roles of residues in the core globular domains are not yet well characterized in any DNA-mediated reactions. To comprehensively understand the functional surface(s) of a core histone, we constructed 320 yeast mutant strains, each of which has a point mutation in a core histone, and identified 42 residues responsible for the suppressor of Ty (Spt(-)) phenotypes, and 8, 30 and 61 residues for sensitivities to 6-azauracil (6AU), hydroxyurea (HU) and methyl-methanesulfonate (MMS), respectively. In addition to residues that affect one specific assay, residues involved in multiple reactions were found, and surprisingly, about half of them were clustered at either the nucleosome entry site, the surface required for nucleosome-nucleosome interactions in crystal packing or their surroundings. This comprehensive mutation approach was proved to be powerful for identification of the functional surfaces of a core histone in a variety of DNA-mediated reactions and could be an effective strategy for characterizing other evolutionarily conserved hub-like factors for which surface structural information is available.
核心组蛋白是核小体的重要组成部分，作为DNA介导反应的全局负调控因子，包括转录、DNA复制和DNA修复。N端尾巴中的修饰残基在转录中已有充分表征，但在DNA复制和DNA修复中并非如此。此外，核心球状结构域中残基在任何DNA介导反应中的作用尚未得到充分表征。为了全面了解核心组蛋白的功能表面，我们构建了320个酵母突变菌株，每个菌株在核心组蛋白中都有一个点突变，并鉴定出42个负责抑制Ty（Spt(-)）表型的残基，以及分别对6-氮尿嘧啶（6AU）、羟基脲（HU）和甲基磺酸甲酯（MMS）敏感的8、30和61个残基。除了影响一种特定检测的残基外，还发现了参与多种反应的残基，令人惊讶的是，其中约一半聚集在核小体进入位点、晶体堆积中核小体-核小体相互作用所需的表面或其周围。这种全面的突变方法被证明对于识别核心组蛋白在各种DNA介导反应中的功能表面很有效，并且可能是表征其他具有表面结构信息的进化保守枢纽样因子的有效策略。
