Deciphering DNA sequence selectivity in V(D)J recombination

破译 V(D)J 重组中的 DNA 序列选择性

• 批准号: 10307113
• 负责人: Karla K Rodgers
• 金额: $ 18.01万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-23 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307113
• 关键词: Adaptive Immune System; Address; Antigen Receptors; B-Lymphocytes; Base Pairing; Binding; Binding Proteins; Biochemical; Biological Assay; Cells; Chromatin Loop; Chromosomal Rearrangement; Code; Complex; Coupled; DNA; DNA Double Strand Break; DNA Repair; DNA Sequence; Data; Development; Elements; Ephrin-A5; Epigenetic Process; Event; Future; Gene Proteins; Genes; Genetic Recombination; Genome; Genome Stability; High-Throughput Nucleotide Sequencing; Immunoglobulins; Individual; Lead; Lymphocyte; Lymphoid; Mediating; Methods; Modification; Nonhomologous DNA End Joining; Nucleosomes; Oncogenic; Outcome; Pathway interactions; Pattern; Peptide Signal Sequences; Plasmids; Positioning Attribute; Proteins; Reaction; Receptor Gene; Risk; Shapes; Site; Specificity; T-Cell Receptor; T-Lymphocyte; Testing; V(D)J Recombination; Variant; base; experimental study; improved; insight; next generation sequencing

Project Summary V(D)J recombination assembles functional antigen receptor genes from component gene segments to produce the diverse repertoire of functional immunoglobulin and T cell receptors in B and T lymphocytes, respectively. RAG1 and RAG2 are lymphoid-specific proteins that catalyze the DNA cleavage steps in V(D)J recombination. RAG-mediated DNA cleavage activity is directed to discrete DNA sequences known as recombination signal sequences (RSSs) that flank the coding gene segments in the antigen receptor loci. In individual recombination reactions, a heterotetrameric RAG1/2 complex binds simultaneously to two RSSs and creates DNA double strand breaks at the border between each RSS and the adjoining coding segment. Joining of the coding segments is carried out by ubiquitous DNA repair factors. Many RSSs are only semi-conserved, such that recombination of poorly conserved RSSs requires promiscuous RAG1/2 activity. RAG1/2 also creates aberrant recombination events at RSS-like sites, called cryptic RSSs (cRSS), located outside of the antigen receptor loci, which can cause oncogenic chromosomal rearrangements. Therefore, RAG1/2 must be promiscuous to facilitate recombination of poorly conserved RSSs, but it must also be precise to avoid off-target cRSSs. To characterize the DNA sequence specificity of RAG1/2, we are developing a high-throughput plasmid recombination method to analyze V(D)J recombination sequence specificity. Greater than 105 extrachromosomal V(D)J recombination substrates of differing sequences are transfected into RAG1/2 expressing cells, the resulting recombination products selectively amplified, and subsequently analyzed by next-generation sequencing. Using this method, we will empirically characterize RSS motifs that enhance RAG1/2 activity to shape a diverse antigen receptor repertoire, as well as identify suboptimal RSS motifs that favor nonconventional V(D)J recombination reactions. To date, highly informative results have been obtained from preliminary studies using this method, which suggest sequence interdependencies exist between different regions of the RSS with significant consequences on the level of V(D)J recombination activity. Furthermore, specific RSS motifs appear to preferentially favor nonconventional V(D)J recombination reactions. Based on our preliminary results, we hypothesize that specific interrelationships within RSSs 1) influence their relative utilization by the RAG proteins and 2) govern their fate in conventional versus aberrant V(D)J recombination reactions. Our aims are to analyze separate regions within the RSS for their effect on V(D)J recombination activity, and second, identify RSS motifs that skew the V(D)J recombination reaction to the formation of aberrant products. Overall, we predict that findings from this project will significantly improve our current understanding of RAG selectivity of RSSs and cRSSs in normal and aberrant V(D)J recombination reactions, respectively.

项目摘要 V（D）J重组从组分基因片段组装功能性抗原受体基因以产生 B和T淋巴细胞中功能性免疫球蛋白和T细胞受体的多样性。 RAG 1和RAG 2是淋巴特异性蛋白，催化V（D）J重组中的DNA切割步骤。 RAG介导的DNA切割活性针对称为重组信号的离散DNA序列 在一个实施方案中，所述抗原受体基因座中的编码基因区段的侧翼包含多个序列（RSS）。在个体重组中 反应中，异源四聚体RAG 1/2复合物同时结合两个RSS并产生DNA双链体。 在每个RSS和相邻编码片段之间的边界处链断裂。编码的加入 片段是由普遍存在的DNA修复因子进行的。许多RSS只是半保守的， 保守性差的RSS的重组需要混杂的RAG 1/2活性。RAG 1/2也会产生异常的 位于抗原受体外的类似RSS位点（称为隐性RSS（cRSS））的重组事件 基因座，这可能会导致致癌染色体重排。因此，RAG 1/2必须混杂， 促进保守性差的RSS的重组，但它也必须是精确的，以避免脱靶cRSS。到 为了表征RAG 1/2的DNA序列特异性，我们正在开发一种高通量质粒， 重组方法分析V（D）J重组序列特异性。大于105 将不同序列的染色体外V（D）J重组底物转染到RAG 1/2中， 表达细胞，选择性扩增所得重组产物，随后通过 下一代测序使用这种方法，我们将经验性地表征RSS图案， RAG 1/2活性，以形成不同的抗原受体库，以及确定次优的RSS基序， 有利于非常规的V（D）J复合反应。迄今为止，已获得了信息量很大的结果 从使用这种方法的初步研究，这表明序列之间存在相互依赖性不同 区域的RSS上的V（D）J重组活动的水平具有显着的后果。此外，委员会认为， 特定的RSS基序似乎优先有利于非常规的V（D）J重组反应。基于 根据我们的初步结果，我们假设RSS中的特定相互关系1）影响它们的相对 RAG蛋白的利用和2）控制它们在常规与异常V（D）J重组中的命运 反应.我们的目标是分析RSS中的不同区域对V（D）J复合的影响 活性，第二，识别使V（D）J重组反应偏向于形成 异常产物总的来说，我们预测，该项目的发现将显着改善我们目前的 了解正常和异常V（D）J重组反应中RSS和cRSS的RAG选择性， 分别