BIOCHEMISTRY AND REGULATION OF V (D) J RECOMBINATION

V (D) J 重组的生物化学和调控

• 批准号: 6510511
• 负责人: Mark S. Schlissel
• 金额: $ 28.3万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510511
• 关键词: B lymphocyte; DNA damage; cell differentiation; gene rearrangement; genetic library; genetically modified animals; immunoglobulin genes; laboratory mouse; nucleosomes; polymerase chain reaction; protooncogene; recombinase; transcription factor

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.

描述（根据研究者的摘要改编）：抗原受体基因是 通过一系列位点特异性DNA从其成分基因段组装 重组反应称为V（d）J重组。基因段 经历该反应的两侧是称为保守的DNA元素 重组信号序列（RSS）。这些元素在 淋巴特异性蛋白Rag1和Rag2的成对时尚 引入一对紧邻RSSS的双链DNA断裂。 然后连接四个结果的DNA末端，形成信号接头和编码 联合的。这些连接步骤利用DSDNA断裂修复的组件 机械在所有细胞中表达。七个复杂的遗传基因座经历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）Alpha，beta，Gamma和Delta基因座。重排 这些基因座的调节是多种调节的：a）Ig基因仅在 仅在T谱系中的B谱系和TCR基因； b）在每个血统中， Ig Mu和TCRβ高度排序抗原受体基因重排 例如，在IG Kappa和TCR Alpha重排之前重排；和 c）单个B或T细胞仅使一个富有成效的（在框架中）重排 在给定的基因座（等位基因排除）。该研究建议的目的是 了解常见的V（d）J重组酶如何识别保守的RSS可以 产生一个基因段重排的精美调节模式。一个 丰富的相关数据导致了一个假设，即可访问性 染色质中重新排列的基因段确定V（d）J的靶向 重组酶。研究人员最近表明重组RAG1和RAG2 补充核提取物时，可以在体外识别并裂解RSSS 在碎布缺陷型淋巴样细胞中的核内。乳沟的模式 对应于核的发育状态，模仿正常 重组酶调节的模式。他继续证明了这一点 定位的单核小体可以防止抹布的裂解和转录 重排基因座内的增强剂对于可访问性和功能至关重要 重组酶。此外，发现核蛋白还 重物组织酶识别和裂解RSS需要需要rag1和rag2 在纯化的基因组DNA底物中。提出了进一步的实验 检查核小体在调节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素，询问是否是否 V（d）J重组酶在体内具有转座酶活性，并确定 RAG2的催化结构域在A中补充淋巴样发育 rag2突变小鼠。