Molecular mechanisms of the RAG recombinase in V(D)J recombination and disease

摘要 脊椎动物免疫的一个标志是抗原受体基因的多样性， 来自通过V（D）J重组的基因编码区段的组合剪接，其切割 和剪接可变（V）、多样性（D）和连接（J）的非连续免疫球蛋白（IG） 基因组中的片段。V（D）J重组中的关键裂解步骤是由 含有多结构域蛋白重组激活基因的淋巴细胞特异性酶 1和2（RAG 1-RAG 2）。RAG重组酶识别特异性重组信号 V、D和J区段的3'端侧翼的RSSs，其由一个或多个RSSs组成。 保守的七聚体、12或23个碱基对的间隔区和保守的九聚体。这些 在间隔区的长度之后，RSS被指定为12-RSS或23-RSS。拼接只能 发生在两侧为12-RSS的一个基因编码区段和两侧为12-RSS的另一个区段之间。 a 23-RSS，确立12/23规则。因为V、D和J段两侧有不同的 RSS如在IgH基因座中，12/23规则有助于确保V、D和 J，但不在同型基因片段内。 RAG复合物催化两个连续反应，切口（链切割）和 发夹形成（链转移），不解离，产生切割的RSS并编码 结束发夹。随后，经典的非同源末端连接（NHEJ）DNA中的蛋白质 修复途径中的基因被募集到RAG复合体中以加工和连接编码片段。 人类RAG突变与一系列遗传性疾病相关， 联合免疫缺陷（SCID）至较轻度变异，如Omenn综合征和RAG 缺乏伴γδ T细胞扩增、肉芽肿形成或母胎植入。异常 V（D）J重组是导致染色体易位的重要机制， 淋巴恶性肿瘤RAG基因被认为只在发育过程中活跃。RAG1 RAG 2再表达通常与自身免疫状态和癌症有关，例如在系统性免疫缺陷综合征中， 红斑狼疮、结直肠癌和结肠癌。在这里，我们提出了一系列 使用冷冻电子显微镜对RAG重组酶进行结构和功能研究， 结晶学从分子水平上了解其功能和调控机制， RAG复合物将有助于理解和潜在的治疗策略， 这些人类疾病。

ABSTRACT A hallmark of vertebrate immunity is the diverse repertoire of antigen-receptor genes that results from combinatorial splicing of gene coding segments by V(D)J recombination, which cleaves and splices variable (V), diversity (D) and joining (J) non-contiguous immunoglobulin (Ig) segments in the genome. The critical cleavage step in V(D)J recombination is executed by the lymphocyte specific enzyme containing the multi-domain proteins recombination-activating gene 1 and 2 (RAG1-RAG2). The RAG recombinase recognizes specific recombination signal sequences (RSSs) flanking the 3' end of the V, D, and J segments, which are composed of a conserved heptamer, a spacer of either 12 or 23 base pairs, and a conserved nonamer. These RSSs are designated as 12-RSS or 23-RSS after the length of the spacer. Splicing can only occur between one gene coding segment flanked by a 12-RSS and another segment flanked by a 23-RSS, establishing the 12/23 rule. Because V, D and J segments are flanked by different RSSs such as in the IgH locus, the 12/23 rule helps to ensure recombination between V, D and J, but not within homotypic gene segments. The RAG complex catalyzes two consecutive reactions, nicking (strand cleavage) and hairpin formation (strand transfer), without dissociation, generating cleaved RSSs and coding end hairpins. Subsequently, proteins in the classical nonhomologous end joining (NHEJ) DNA repair pathway are recruited to the RAG complex to process and join the coding segments. Human RAG mutations are associated with a spectrum of genetic disorders ranging from severe combined immunodeficiency (SCID) to milder variants, such as Omenn syndrome and RAG deficiency with γδ T cell expansion, granuloma formation, or maternofetal engraftment. Aberrant V(D)J recombination is an important mechanism responsible for chromosomal translocations in lymphoid malignancies. RAG genes are supposed to be active only during development. RAG1 and RAG2 re-expression is often linked to autoimmune states and cancers, such as in systemic lupus erythematosus, colorectal cancer and colon cancer. Here we propose a series of structural and functional studies on the RAG recombinase using cryo-electron microscopy and crystallography. A molecular understanding on the functions and regulatory mechanisms of the RAG complex will contribute to the understanding and the potential therapeutic strategies for these human diseases.