RAG-2 Promotes Heptamer Occupancy by RAG-1 in the Assembly of a V(D)J Initiation Complex
RAG-2 Promotes Heptamer Occupancy by RAG-1 in the Assembly of a V(D)J Initiation Complex
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DOI：
10.1128/mcb.19.5.3674
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发表时间：
1999-05
影响因子：
5.3
通讯作者：
P. Swanson;S. Desiderio
中科院分区：
文献类型：
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作者：
P. Swanson;S. Desiderio
摘要 V(D)J 重组发生在含有保守七聚体和九聚体元件的重组信号序列 (RSS) 处。 RAG-1 和 RAG-2 通过在七聚体和抗原受体编码片段之间切割 DNA 来启动重组。 RAG-1 单独接触九聚体，但与七聚体的相互作用微弱（如果有的话）。 RAG-2 本身没有 DNA 结合活性，但在 RAG-1 存在的情况下促进七聚体占据；人们对 RAG-2 如何与 RAG-1 协作知之甚少。在这里，我们检查了 RAG-RSS 复合物的组成以及 RAG-1 和 RAG-2 对七聚体结合的相对贡献。 RAG-1 以二聚体形式存在于复合物中，并带有带有 12 bp 间隔区的分离 RSS，无论 RAG-2 是否存在；然而，RAG-1 只有一个亚基参与九聚体结合。相反，在含有两种 RAG 蛋白的复合物中，通过电泳迁移率变动测定无法检测到多聚体 RAG-2。 DNA-蛋白质光交联表明七聚体接触虽然被 RAG-2 增强，但主要由 RAG-1 介导。 RAG-2 交联虽然效率低于 RAG-1，但在七聚体编码连接处附近可检测到。这些观察结果提供了证据，表明 RAG-2 改变了 RAG-1 的构象或方向，从而稳定了 RAG-1 与七聚体的相互作用，并表明两种蛋白质在切割位点附近与 RSS 相互作用。
ABSTRACT V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.