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

RAG重组酶在V(D)J重组和疾病中的分子机制

• 批准号: 9159111
• 负责人: Hao Wu
• 金额: $ 65.37万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9159111
• 关键词: Antigen Receptors; Autoimmune Process; Autoimmunity; Base Pairing; Binding; Biochemical; Biological; Boston; Catalysis; Chemicals; Chromosomal translocation; Cleaved cell; Code; Collaborations; Colon Carcinoma; Colorectal Cancer; Complex; Cryoelectron Microscopy; Crystallization; DNA Binding; DNA Double Strand Break; DNA Repair Pathway; Development; Disease; Dissociation; Engineering; Engraftment; Ensure; Enzymes; Gene Mutation; Genes; Genetic Recombination; Genome; Genomic Instability; Granuloma; HMGB1 gene; Hereditary Disease; High Mobility Group Proteins; Human; IGH@ gene cluster; Immunity; Immunoglobulins; Immunologic Deficiency Syndromes; In Vitro; Length; Link; Lymphocyte; Malignant Neoplasms; Malignant lymphoid neoplasm; Mediating; Molecular; Molecular Conformation; Pediatric Hospitals; Peptide Signal Sequences; Play; Process; Proteins; RAG1 gene; RNA Splicing; Reaction; Receptor Gene; Recruitment Activity; Resolution; Role; Series; Severe Combined Immunodeficiency; Signal Transduction; Spliced Genes; Structure; Synapses; Syndrome; Systemic Lupus Erythematosus; T-Lymphocyte; Tertiary Protein Structure; Therapeutic; Time; V(D)J Recombination; Variant; Zebrafish; combinatorial; dimer; electron crystallography; gene function; human disease; insight; medical schools; recombinase; reconstitution; single molecule; success

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.

摘要 脊椎动物免疫的一个标志是抗原受体基因的多样化，导致 通过V(D)J重组对基因编码片段进行组合剪接，从而切割 和剪接可变(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重组中的关键切割步骤由 含有多结构域蛋白重组激活基因的淋巴细胞特异酶 1和2(RAG1-RAG2)。RAG重组酶识别特定重组信号 位于V、D和J片段3‘端两侧的序列(RSS)，由 保守的七聚体，一个由12或23个碱基对组成的间隔区，以及一个保守的九聚体。这些 在间隔物长度之后，RSS被指定为12-RS或23-RS。拼接只能 发生在一个由12-RSS侧翼的基因编码片段和另一个侧翼为 23-RSS，建立了12/23规则。因为V、D和J段两侧有不同的 RSS，如在IgH基因座，12/23规则有助于确保V、D和 J，但不在同型基因片段内。 RAG复合体催化两个连续的反应，即切割(链切割)和 发夹形成(链转移)，不解离，产生切割的RSS和编码 发夹末端。随后，经典的非同源末端连接(NHEJ)DNA中的蛋白质 修复途径被招募到RAG复合体，以处理和连接编码片段。 人类RAG突变与一系列遗传性疾病有关，从严重的 联合免疫缺陷(SCID)对较轻的变种，如Omenn综合征和RAG γδT细胞缺乏，肉芽肿形成，或母胎植入。反常的 V(D)J重组是染色体易位的重要机制 淋巴样恶性肿瘤。RAG基因被认为只在发育过程中活跃。RAG1 RAG2的重新表达通常与自身免疫状态和癌症有关，例如在系统性红斑狼疮中 红斑狼疮、结直肠癌和结肠癌。在这里，我们提出了一系列 用冷冻电子显微镜和电子显微镜研究RAG重组酶的结构和功能 结晶学。细胞外信号转运蛋白的功能和调控机制的分子理解 RAG复合体将有助于理解和潜在的治疗策略 这些人类疾病。