Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro
Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro
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DOI：
10.1074/jbc.m503063200
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发表时间：
2005-09-02
影响因子：
4.8
通讯作者：
Swanson, PC
中科院分区：
文献类型：
--
作者：
Bergeron, S;Madathiparambil, T;Swanson, PC
RAG-1和RAG-2通过12/23对V(D)J重组信号序列的突触和裂解启动V(D)J重组。RAG-RSS复合体的体外组装和活性受到以HMGB1为代表的HMG-box家族高迁移率基团蛋白的促进。HMGB1如何刺激RAG复合体的DNA结合和切割活性仍不清楚。HMGB1含有两个同源的HMG-box DNA结合域，称为A和B，由一段碱性残基连接到一个高度酸性的C-末端。为了确定刺激RAG介导的RSS结合和切割所需的HMGB1的决定因素，我们制备了一组突变的HMGB1蛋白，并测试了它们增强RAG介导的RSS结合和切割活性的能力。结合使用迁移率改变和凝胶内切割分析，我们发现HMGB1主要通过B盒的活性促进RAG介导的切割，但最佳刺激需要一个功能A盒拴在正确的方向上。A或B盒突变体不能促进RAG突触复合体的形成，但当这些突变体去掉酸性尾巴时，这一缺陷就会得到缓解。
RAG-1 and RAG-2 initiate V(D) J recombination through synapsis and cleavage of a 12/23 pair of V( D) J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.