Biochemistry and Regulation of V(D)J Recombination

描述(申请人提供)：编码免疫球蛋白和T细胞受体分子的基因由V、D和J基因片段通过一系列称为V(D)J重组的新型定点DNA重组反应组装而成。重组酶由淋巴细胞特异性蛋白RAG1和RAG2以及广泛表达的非同源末端连接蛋白组成。重组酶识别基因片段两侧的保守重组信号序列(RSS)，并引入dsDNA断裂，这些断裂被修复形成编码和信号关节。B细胞和T细胞中7个复杂抗原受体基因座的重组受谱系特异性、谱系内顺序和等位基因排除的调节。我们和其他人已经证明，V(D)J重组在一定程度上受到染色质结构中RSS的可及性的调节，而未重排基因片段的胚系转录反映或实际上导致了染色质的可及性。这一建议的中心目标是在生化水平上了解V(D)J重组的生物调控机制。这一目标将通过追求以下具体目标来实现：1)确定跨J-kappa基因片段簇的核小体的位置和结构，以及核小体如何被破坏以允许重组酶的可及性；2)确定V(D)J重组酶是否在体内损伤DNA，以及从缺失到断裂的DNA转换是否是反应中的调节步骤；3)确定RAG2的核心域突变体在体内的催化性质；以及4)确定转录调控元件在调节V(D)J重组中的作用。这项工作意义重大，因为抗原受体在获得性免疫中的重要作用，以及重组酶可能参与与淋巴系统恶性肿瘤相关的染色体易位的产生。

DESCRIPTION (provided by applicant): The genes which encode immunoglobulin and T cell receptor molecules are assembled from V, D, and J gene-segments by a novel series of site-specific DNA recombination reactions known as V(D)J recombination. The recombinase consists of the lymphocyte-specific proteins RAG1 and RAG2 and the widely expressed non-homologous end-joining proteins. The recombinase recognizes conserved recombination signal sequences (RSSs) which flank gene segments and introduces dsDNA breaks which are repaired to form coding and signal joints. Recombination at the seven complex antigen-receptor loci in B and T cells is regulated with respect to lineage specificity, order within a lineage, and allelic exclusion. We and others have shown that V(D)J recombination is regulated in part by the accessibility of RSSs within chromatin structure and that germline transcription of unrearranged gene segments either reflects or actually causes chromatin accessibility. The central goal of this proposal is to understand, at the biochemical level, the mechanisms which dictate the biological regulation of V(D)J recombination. This goal will be pursued through pursuit of the following specific aims: 1) to determine the location and structure of nucleosomes across the J-kappa cluster of gene segments and how nucleosomes are disrupted to allow recombinase accessibility; 2) to determine whether the V(D)J recombinase nicks DNA in vivo and whether the conversion from nicked to broken DNA is a regulated step in the reaction; 3) to determine the catalytic properties in vivo of a core-domain mutant of RAG2; and 4) to define the role of transcriptional regulatory elements in the regulation of V(D)J recombination. This work is significant because of the essential role of antigen receptors in acquired immunity and the potential involvement of the recombinase in generation of chromosomal translocations associated with lymphoid malignancies.