Biochemistry and Regulation of V(D)J Recombination

V(D)J 重组的生物化学和调控

• 批准号: 7433339
• 负责人: Mark S. Schlissel
• 金额: $ 34.3万
• 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 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7433339
• 关键词: Antigen Receptors; Antigens; B-Lymphocytes; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Cells; Chromatin; Chromatin Structure; Chromosomal translocation; Chromosome Pairing; Cleaved cell; Code; Complex; DNA; DNA Sequence Rearrangement; Development; Developmental Biology; Disruption; Elements; Enhancers; Event; Excision; Exclusion; Funding; Gene Cluster; Gene Components; Gene Rearrangement; Generations; Genes; Genetic; Genetic Recombination; Genetic Transcription; Goals; Immunoglobulins; In Vitro; J segment gene; Joints; Location; Lymphocyte; Malignant lymphoid neoplasm; Maps; Mus; Nonhomologous DNA End Joining; Nucleosomes; Peptide Signal Sequences; Plasmids; Play; Positioning Attribute; Property; Proteins; Proto-Oncogenes; Reaction; Receptor Gene; Regulation; Regulator Genes; Reporter; Research Personnel; Role; Series; Signal Transduction; Site; Specificity; Spectrum Analysis; Staging; Structure; Surface; Synapses; T-Cell Receptor; T-Lymphocyte; TCF3 gene; Techniques; Testing; V(D)J Recombination; VDJ Recombinases; Work; acquired immunity; dimer; in vivo; mutant; novel; programs; promoter; recombinase; repaired; research study; transcription factor

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.

描述（由申请人提供）：编码免疫球蛋白和T细胞受体分子的基因是通过V，D和J基因分析组装的，这是一系列新型位点特异性DNA重组反应，称为V（d）J重组。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理末端结合蛋白组成。重组酶识别侧翼基因片段并引入DSDNA断裂的保守重组信号序列（RSS），这些断裂已修复以形成编码和信号关节。 B和T细胞中七个复合物抗原受体基因座的重组对谱系特异性，谱系内的顺序和等位基因排除受到调节。我们和其他人表明，V（d）J重组部分受染色质结构中RSS的可及性的一部分调节，并且透明基因片段的种系转录要么反映或实际引起染色质的可及性。该提案的核心目标是在生化层面上了解决定V（d）J重组的生物学调节的机制。将通过追求以下特定目的来实现此目标：1）确定基因段的J-Kappa群中的核小体的位置和结构，以及如何破坏核小体以允许重组酶可及性； 2）确定V（d）J重组酶在体内刻痕DNA以及从划痕到断裂DNA的转化是否是反应中的调节步骤； 3）确定rag2的核心域突变体体内的催化特性； 4）定义转录调节元件在V（d）J重组调节中的作用。这项工作很重要，因为抗原受体在获得的免疫力中的重要作用以及重组酶在与淋巴恶性肿瘤相关的染色体易位产生中的潜在参与。