Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein
Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein
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DOI：
10.1074/jbc.m502155200
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发表时间：
2005-08-26
影响因子：
4.8
通讯作者：
Thelen, MP
中科院分区：
文献类型：
--
作者：
Beernink, PT;Hwang, M;Thelen, MP
对 DNA 修复和细胞周期控制系统至关重要的蛋白质相互作用通常由属于结构保守 BRCT 结构域超家族的模块协调。由于 BRCT 相互作用的机制及其意义尚不清楚，我们试图定义那些协调碱基切除修复和单链断裂修复的 BRCT 模块的亲和力和特异性。这些途径的共同点是必需的 XRCC1 DNA 修复蛋白，它与至少九种其他蛋白和 DNA 相互作用。在这里，我们表征了四个纯化 BRCT 结构域的相互作用，其中两个来自 XRCC1，它们的两个伙伴来自 DNA 连接酶 III α 和聚（ADP-核糖基）聚合酶 1。选择了识别连接酶 III α BRCT 结构域但不识别其他 BRCT 结构域的单克隆抗体，并用于捕获相关的连接酶 III α BRCT 复合物。为了检查分离的 BRCT 结构域和成对结构域复合物的组装状态，我们使用了尺寸排阻色谱法与在线光散射相结合。该分析表明，分离的 BRCT 结构域形成同源寡聚体，并且 C 端 XRCC1 结构域和连接酶 III α 结构域之间的 BRCT 复合物是具有 2:2 化学计量的异四聚体。使用亲和捕获和表面等离子共振方法，我们确定在同源 BRCT 结构域对之间发生具有高纳摩尔解离常数的特定异聚相互作用。提出了 XRCC1(.)DNA 连接酶 III α 异四聚体的结构模型作为核心碱基切除修复复合物，其构成了其他修复蛋白和 DNA 接近的高阶复合物的支架。
Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase III alpha and poly( ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase III alpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase III alpha BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase III alpha domain is a heterotetramer with 2: 2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1(.)DNA ligase III alpha heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.