Deciphering DNA sequence selectivity in V(D)J recombination

项目摘要 V(D)J重组将组成基因片段中的功能性抗原受体基因组装成 B和T淋巴细胞的功能免疫球蛋白和T细胞受体的不同谱系。 RAG1和RAG2是淋巴特异性蛋白，在V(D)J重组中催化DNA裂解步骤。 RAG介导的DNA切割活性直接作用于离散的DNA序列，称为重组信号 位于抗原受体基因座编码基因片段两侧的序列(RSS)。在个体重组中 反应中，一个异四聚体RAG1/2复合体同时与两个RSS结合并产生DNA双链 链在每个RSS和相邻编码片段之间的边界处断裂。编码的接合 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，因此 保守程度较低的RSS的重组需要混杂的RAG1/2活性。RAG1/2也会造成反常 位于抗原受体外部的RSS样位点上的重组事件，称为隐蔽RSS(CRSS) 可导致致癌染色体重排的基因座。因此，RAG1/2必须是混杂的 促进保守程度较低的RSS的重组，但也必须精确，以避免偏离靶标的cRSS。至 鉴定RAG1/2的DNA序列特异性，我们正在开发一种高通量的质粒 重组法分析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重组底物导入RAG1/2 表达细胞，得到的重组产物选择性地扩增，随后通过 下一代测序。使用这种方法，我们将经验地表征增强的RSS主题 RAG1/2活性，以形成不同的抗原受体谱系，以及确定次优的RSS基序 有利于非常规的V(D)J复合反应。到目前为止，已经取得了信息量很大的结果 使用这种方法的初步研究表明，不同的基因之间存在序列相互依赖 对V(D)J重组活性水平有显著影响的RSS区域。此外， 特定的RSS基序似乎优先支持非传统的V(D)J重组反应。基于 根据我们的初步结果，我们假设RSS中的特定相互关系会影响它们的相对关系 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重组的影响 第二，确定扭曲V(D)J重组反应形成的RSS基序 异常产品。总体而言，我们预测该项目的结果将显著改善我们目前的 了解RSSs和cRSSs在正常和异常V(D)J复合反应中的RAG选择性， 分别进行了分析。

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.