BIOCHEMISTRY AND REGULATION OF V (D) J RECOMBINATION

描述（改编自研究者摘要）：抗原受体基因是 由一系列位点特异性DNA组成的基因片段 这种复合反应称为V（D）J复合。基因区段 在进行这种反应时，两侧是保守的DNA元件， 重组信号序列（RSS）。这些元素在一个 淋巴特异性蛋白RAG 1和RAG 2成对表达， 引入紧邻RSS的一对双链DNA断裂。 然后将四个所得DNA末端连接以形成信号接头和编码接头。 关节这些连接步骤利用dsDNA断裂修复的组分 在所有细胞中表达的机器。7个复杂遗传位点经历V（D）J 包括免疫球蛋白（IG）μ、κ和λ基因座的重组，以及 T细胞受体（TCR）α、β、γ和δ基因座。重排 这些基因座的调控方式有几种：a）IG基因完全重排，只有在 B谱系和仅在T谱系中的TCR基因; B）在每个谱系内， 抗原受体基因重排是高度有序的，其中IG mu和TCR β 例如，在IG κ和TCR α重排之前重排;和 c）单个B或T细胞仅进行一次有效（框内）重排 等位基因排斥（allelic exclusion）这项研究计划的目的是 理解识别保守RSS的常见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 大量的相关数据导致了一个假设， 染色质中基因片段的重排决定了V（D）J的靶向 重组酶研究人员最近表明，重组RAG 1和RAG 2 当添加核提取物时，可以在体外识别和切割RSS 在RAG缺陷淋巴细胞的细胞核内。卵裂的模式 对应于细胞核的发育状态，模仿正常的 重组酶的调节模式。他接着指出， 定位的单核小体可以阻止RAG切割， 重排位点内的增强子对于可及性和功能至关重要 重组酶此外，还发现核蛋白除了 RAG 1和RAG 2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。提出实验以进一步 检查核小体在调节V（D）J重组中的作用，以纯化 和分子克隆因子，帮助靶向重组， 所述V（D）J重组酶在体内具有转座酶活性，并且为了确定所述V（D）J重组酶的活性， RAG 2的催化结构域补充淋巴发育的能力， RAG 2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.