BIOCHEMISTRY AND REGULATION OF V (D) J RECOMBINATION

描述(改编自研究人员摘要)：抗原受体基因 由它们的组成基因片段由一系列位点特异性DNA组装而成 复合反应称为V(D)J复合。基因片段 在这个反应的侧翼是保守的DNA元件，称为 重组信号序列(RSS)。这些元素在 通过淋巴特异性蛋白RAG1和RAG2的配对方式， 引入一对紧邻RSS的双链DNA断裂。 然后将生成的四个DNA末端连接起来，形成一个信号接头和一个编码 联合。这些连接步骤利用dsDNA断裂修复的组件 在所有细胞中表达的机械。7个复杂遗传基因座发生V(D)J 重组包括免疫球蛋白(Ig)Mu、kappa和lambda基因座以及 T细胞受体(TCR)的阿尔法、贝塔、伽马和德尔塔基因座。重新安排 这些基因座中有几种调节方式：a)免疫球蛋白基因仅在 B谱系和TCR基因仅在T谱系中；b)在每个谱系中， 抗原受体基因重排高度有序，Ig Mu和TCRβ 例如，在Ig kappa和TCRα重排之前的重排；以及 C)单个B或T细胞只进行一次有效的(帧内)重排 在给定的基因座上(等位基因排除)。这项研究提案的目的是 了解识别保守RSS的普通V(D)J重组酶如何 产生一种精确调控的基因片段重排模式。一个 大量的相关数据导致了这样一种假设，即 染色质中基因片段的重新排列决定了V(D)J的靶向性 重组酶。研究人员最近发现，重组RAG1和RAG2 当添加核提取物时，可在体外识别和切割RSS 在来自RAG缺陷的淋巴样细胞的细胞核内。乳沟的模式 与核的发育状态相对应，模仿正常的 重组酶的调控模式。他继续展示了这一点 定位的单核小体可以防止RAG裂解和转录 重新排列基因座内的增强剂对可及性和功能至关重要 重组酶的活性。此外，还发现除了核蛋白外， RAG1和RAG2是重组酶识别和切割RSS所必需的 在纯化的基因组DNA底物中。建议进行实验，以进一步 检测核小体在调节V(D)J重组中的作用，以纯化 以及有助于靶向重组的分子克隆因子，以询问是否 V(D)J重组酶在体内具有转座酶活性，并测定 RAG2催化结构域在补体淋巴发育中的作用 RAG2突变小鼠。

DESCRIPTION (adapted from investigator's abstract): Antigen receptor genes are assembled from their component gene segments by a series of site-specific DNA recombination reactions known as V(D)J recombination. Gene segments which undergo this reaction are flanked by conserved DNA elements called recombination signal sequences (RSSs). These elements are recognized in a pairwise fashion by the lymphoid-specific proteins, RAG1 and RAG2, which introduce a pair of double-strand DNA breaks immediately adjacent to the RSSs. The four resultant DNA ends are then joined to form a signal joint and a coding joint. These joining steps utilize components of the dsDNA break repair machinery expressed in all cells. Seven complex genetic loci undergo V(D)J recombination including the immunoglobulin (Ig) mu, kappa, and lambda loci and the T cell receptor (TCR) alpha, beta, gamma, and delta loci. The rearrangement of these loci is regulated in several ways: a) Ig genes fully rearrange only in the B lineage and TCR genes only in the T lineage; b) within each lineage, antigen receptor gene rearrangement is highly ordered, with Ig mu and TCR beta rearrangement preceding Ig kappa and TCR alpha rearrangement for example; and c) an individual B or T cell makes only one productive (in frame) rearrangement at a given locus (allelic exclusion). The aim of this research proposal is to understand how a common V(D)J recombinase recognizing a conserved RSS can generate an exquisitely regulated pattern of gene-segment rearrangement. A wealth of correlative data has led to the hypothesis that accessibility of rearranging gene segments in chromatin determines the targeting of the V(D)J recombinase. The investigator showed recently that recombinant RAG1 and RAG2 when supplemented with nuclear extract could recognize and cleave RSSs in vitro within nuclei from RAG-deficient lymphoid cells. The pattern of cleavage corresponds to the state of development of the nuclei, mimicking the normal pattern of regulation of the recombinase. He went on to show that precisely positioned mononucleosomes can prevent RAG cleavage and that transcriptional enhancers within rearranging loci were critical for accessibility and function of the recombinase. In addition, it was found that nuclear proteins in addition to RAG1 and RAG2 were required for the recombinase to recognize and cleave RSSs within purified genomic DNA substrates. Experiments are proposed to further examine the role of nucleosomes in regulating V(D)J recombination, to purify and molecularly clone factors which help target recombination, to ask whether the V(D)J recombinase has transposase activity in vivo, and to determine the ability of the catalytic domain of RAG2 to complement lymphoid development in a RAG2 mutant mouse.