Mismatch repair in V region mutation and isotype switching

V区突变和同种型转换中的错配修复

• 批准号: 8900937
• 负责人: MATTHEW D SCHARFF
• 金额: $ 3.13万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8900937
• 关键词: ATP phosphohydrolase; Affect; Affinity; Affinity Chromatography; Antibodies; Antibody Diversity; Antigens; B-Lymphocytes; Base Excision Repairs; Binding; Cancer Patient; Cells; Characteristics; Chromatin; Chromatin Remodeling Factor; Complementary DNA; Complex; Core Protein; DNA Binding Domain; DNA Double Strand Break; DNA Sequence; Data; EXO1 exonuclease; EXO1 gene; Event; Exonuclease; Frequencies; Genes; Genetic; Genetic Recombination; Genome Stability; HTATIP gene; Health; Hot Spot; ISWI; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Immunoprecipitation; Infection; Inherited; Knock-in Mouse; Knockout Mice; Lead; MLH1 gene; MSH2 gene; MSH6 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mismatch Repair; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; Organism; PMS2 gene; PWWP Domain; Pathway interactions; Pattern; Phenotype; Process; Proteins; Proteomics; Proto-Oncogenes; Recruitment Activity; Regulation; Repair Complex; Replication-Associated Process; Resolution; Role; SMARCA5 gene; Site; Somatic Mutation; Spleen; Structure; Structure of germinal center of lymph node; T-Lymphocyte; Tissues; Western Blotting; activation-induced cytidine deaminase; base; chromatin remodeling; deep sequencing; histone modification; knock-down; mutant; null mutation; oxidative damage; pathogen; pol Gene Products; protein protein interaction; repaired; research study; scaffold; small hairpin RNA; stoichiometry; transition mutation

DESCRIPTION (provided by applicant): The immunoglobulin (Ig) genes in germinal center B cells undergo a high rate of somatic mutation and recombination to achieve the affinity maturation and isotype switching that generates protective antibodies. These processes are initiated by activation induced deaminase protein (AID) which generates G:U mismatches that are processed by replication, base excision repair and mismatch repair (MMR) to produce the mutations required for somatic hypermutation of antibody variable and switch regions. MMR is responsible for ~50% of these mutations most of which are in A:T bases. While canonical MMR normally maintains the integrity and stability of the genome, at the Ig gene it appears to have shifted to an errorprone non-canonical pathway of MMR, which mediates extensive mutation and recombination. Here we propose to examine how B cells regulate and recruit error prone MMR to immunoglobulin genes by examining new genetically modified mice with separation-of-function mutations: 1) the Msh6E433A mutation in the DNA binding domain of MSH6 is at the site that recognizes and binds the mismatched base and is defective in repairing oxidative damage through the non-canonical pathway of MMR; 2) the Msh6S144I mutation in the PWWP domain of MSH6 was identified in hereditary non-polyposis cancer (HNPCC) patients and is defective in binding the H3K36me3 histone modification and presumably in recruiting MSH6 to chromatin; and 3) the Msh2G674D and Msh6T1217D mice carry ATPase mutants that model other HNPCC mutations and are defective in undergoing conformational changes required to recruit downstream factors. We will use deep sequencing of the Ig variable and switch regions of the mutant mice to determine the impact of these mutations in MMR on somatic hypermutation and class switch recombination. This will provide a high-resolution phenotype of each of the MMR mutations. We will seek the mechanisms responsible for these phenotypes by determining the stoichiometry and interactions of the core MMR proteins in wild type and mutant B cells. To accomplish this in primary B cells, we will carry out immunoprecipitation (IP), tandem affinity purification (TAP), western blot analysis and mass spectrometry using mice expressing TAP-tagged MSH6 and EXO1 in their endogenous loci. Finally we will analyze the role of chromatin remodeling by examining the role of chromatin remodeling proteins that we have found to inhibit CSR in an shRNA screen of CH12F3 cells and that are part of the MMR interactome. We believe that dissecting out the role of each of the domains of MSH6 and of MSH2 and identifying the differences in the MMR complexes and chromatin remodeling factors responsible for V region mutation and class switch recombination will reveal how error prone MMR is largely restricted to the Ig gene and ultimately how changes in the regulation of these processes leads to cancer in many tissues.

描述（由申请人提供）：生发中心B细胞中的免疫球蛋白（Ig）基因经历了高的体细胞突变和重组率，以实现产生保护性抗体的亲和力成熟和同型转换。这些过程是通过激活诱导的脱氨酶蛋白（AID）引发的，该蛋白（AID）产生了通过复制，基础切除修复和不匹配修复（MMR）处理的G：U不匹配，以产生抗体变量和开关区域的体躯体过度符号所需的突变。 MMR负责这些突变中的约50％，其中大多数位于A：T碱基中。虽然规范MMR通常保持基因组的完整性和稳定性，但在IG基因上，它似乎已转移到MMR的错误非传统途径，该途径介导了广泛的突变和重组。在这里，我们建议检查B细胞如何通过检查具有功能分离突变的新的遗传改性小鼠来调节和募集误差，可容易受到免疫球蛋白基因的影响：1）MSH6E4333a在MSH6的DNA结合结构域中的MSH6E433A突变是在MSH6的位置识别并通过基础构成氧化的氧化和氧化为氧化的氧化和氧化为氧化的氧化。 2）在遗传非型癌（HNPCC）患者中鉴定出MSH6的PWWP结构域中的MSH6S144I突变，并且在结合H3K36ME3组蛋白修饰并促进MSH6与染色质的H3K36ME3组蛋白修饰方面有缺陷； 3）MSH2G674D和MSH6T1217D小鼠携带ATPase突变体，该突变体对其他HNPCC突变进行建模，并且在募集下游因子所需的构象变化方面存在缺陷。我们将使用突变小鼠的IG变量和开关区域的深层测序，以确定这些突变对MMR对体太突变和类开关重组的影响。这将提供每个MMR突变的高分辨率表型。我们将通过确定野生型和突变B细胞中核心MMR蛋白的化学计量和相互作用来寻求负责这些表型的机制。为了在原代B细胞中实现这一目标，我们将使用在其内源性基因座中表达TAP的MSH6和EXO1的小鼠进行免疫沉淀（IP），串联亲和力纯化（TAP），Western blot分析和质谱法。最后，我们将通过检查染色质重塑蛋白的作用来分析染色质重塑的作用，这些蛋白质在CH12F3细胞的shRNA筛选中抑制了CSR，并且是MMR相互作用组的一部分。我们认为，剖析MSH6和MSH2的每个领域的作用，并确定负责V区域突变和类转换重组的MMR复合物和染色质重塑因子的差异，将在很大程度上揭示错误的MMR在很大程度上限制在IG基因上，并最终限制了这些过程中如何将这些过程的变化导致许多组织中的癌症。