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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的脊椎动物免疫的标志是导致抗原受体基因的潜水从v（d）J重组的基因编码段的组合剪接，裂解和香料变量（V），多样性（D）和连接（J）非连续免疫球蛋白（IG）基因组中的段。 V（d）J重组中的关键裂解步骤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序列（RSS）在V，D和J段的3'端的两侧，由A组成保守的Hepater，12或23个碱基对的垫片和一个保守的非照明器。这些 RSS被指定为垫片长度后的12-RSS或23-RS。剪接只能发生在一个基因编码段之间，两侧是12-RSS和另一个段的段 23-RS，建立12/23规则。因为V，D和J段是不同的诸如IGH基因座之类的RSS，12/23规则有助于确保V，D和 J，但不在同型基因段中。抹布复合物催化了两种连续的反应，划痕（链裂解）和发夹形成（链转移），无解离，产生切割的RSS和编码结束发夹。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抹布综合体以处理并加入编码段。人抹布突变与一系列遗传疾病有关与米勒变种的合并免疫缺陷（SCID），例如Omenn综合征和抹布缺乏γδT细胞膨胀，肉芽肿或母体植入的缺乏。异常 V（d）J重组是负责染色体易位的重要机制淋巴恶性肿瘤。抹布基因只有在发育过程中活跃。 rag1 RAG2重新表达通常与自身免疫状态和癌症有关，例如红斑狼疮，有色癌和结肠癌。在这里，我们提出了一系列使用冷冻电子显微镜和晶体学。分子理解对功能和调节机制的理解抹布综合体将有助于理解和潜在的理论策略这些人类疾病。