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，其结果是 通过 V(D)J 重组对基因编码片段进行组合剪接，从而切割 和剪接变量 (V)、多样性 (D) 和连接 (J) 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 含有多域蛋白重组激活基因的淋巴细胞特异性酶 1和2(RAG1-RAG2)。 RAG重组酶识别特定的重组信号 V、D 和 J 片段 3' 端侧翼的序列 (RSS)，由 保守的七聚体、12 或 23 个碱基对的间隔区以及保守的九聚体。这些 RSS 在间隔符的长度之后被指定为 12-RSS 或 23-RSS。拼接只能 发生在侧翼为 12-RSS 的一个基因编码片段和侧翼为 12-RSS 的另一基因编码片段之间 23-RSS，建立 12/23 规则。因为 V、D 和 J 段的两侧是不同的 RSS，例如在 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的反应：切口（链断裂）和 发夹形成（链转移），无需解离，生成切割的 RSS 和编码 末端发夹。随后，经典非同源末端连接 (NHEJ) DNA 中的蛋白质 修复途径被招募到 RAG 复合体来处理和连接编码片段。 人类 RAG 突变与一系列遗传性疾病相关，从严重到严重 联合免疫缺陷 (SCID) 至较轻微的变异，例如 Omenn 综合征和 RAG γδ T 细胞扩增、肉芽肿形成或母胎植入缺陷。异常的 V(D)J重组是染色体易位的重要机制 淋巴系统恶性肿瘤。 RAG 基因应该只在发育过程中活跃。 RAG1 RAG2 重新表达通常与自身免疫状态和癌症有关，例如在全身性癌症中 红斑狼疮、结直肠癌和结肠癌。在这里我们提出一系列 使用冷冻电子显微镜对 RAG 重组酶进行结构和功能研究 晶体学。对功能和调节机制的分子理解 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.