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
基本信息
- 批准号:8900937
- 负责人:
- 金额:$ 3.13万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ATP phosphohydrolaseAffectAffinityAffinity ChromatographyAntibodiesAntibody DiversityAntigensB-LymphocytesBase Excision RepairsBindingCancer PatientCellsCharacteristicsChromatinChromatin Remodeling FactorComplementary DNAComplexCore ProteinDNA Binding DomainDNA Double Strand BreakDNA SequenceDataEXO1 exonucleaseEXO1 geneEventExonucleaseFrequenciesGenesGeneticGenetic RecombinationGenome StabilityHTATIP geneHealthHot SpotISWIImmunoglobulin Class SwitchingImmunoglobulin GenesImmunoglobulin Somatic HypermutationImmunoglobulin Switch RecombinationImmunoglobulinsImmunoprecipitationInfectionInheritedKnock-in MouseKnockout MiceLeadMLH1 geneMSH2 geneMSH6 geneMalignant NeoplasmsMass Spectrum AnalysisMediatingMismatch RepairModelingMolecularMusMutant Strains MiceMutateMutationOrganismPMS2 genePWWP DomainPathway interactionsPatternPhenotypeProcessProteinsProteomicsProto-OncogenesRecruitment ActivityRegulationRepair ComplexReplication-Associated ProcessResolutionRoleSMARCA5 geneSiteSomatic MutationSpleenStructureStructure of germinal center of lymph nodeT-LymphocyteTissuesWestern Blottingactivation-induced cytidine deaminasebasechromatin remodelingdeep sequencinghistone modificationknock-downmutantnull mutationoxidative damagepathogenpol Gene Productsprotein protein interactionrepairedresearch studyscaffoldsmall hairpin RNAstoichiometrytransition mutation
项目摘要
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(申请人提供):生发中心B细胞中的免疫球蛋白(Ig)基因经历高速率的体细胞突变和重组,以实现亲和成熟和同型转换,从而产生保护性抗体。这些过程是由激活诱导脱氨酶蛋白(AID)启动的,它产生G:U错配,通过复制、碱基切除修复和错配修复(MMR)处理,产生抗体可变区和开关区体细胞超突变所需的突变。这些突变中约有50%是由MMR引起的,其中大多数是在A:T碱基中。虽然典型MMR通常维持基因组的完整性和稳定性,但在Ig基因上,它似乎已经转移到易出错的非典型MMR途径,介导广泛的突变和重组。在这里,我们提出通过检测具有功能分离突变的新转基因小鼠来研究B细胞如何调节和招募易出错的MMR到免疫球蛋白基因:1)MSH6 DNA结合域的Msh6E433A突变位于识别和结合错配碱基的位点,并且在通过MMR的非规范途径修复氧化损伤方面存在缺陷;2)在遗传性非息肉病癌(HNPCC)患者中发现了MSH6 PWWP结构域的Msh6S144I突变,该突变在结合H3K36me3组蛋白修饰方面存在缺陷,可能在将MSH6募集到染色质上;3) Msh2G674D和Msh6T1217D小鼠携带的ATPase突变可以模拟其他HNPCC突变,并且在进行招募下游因子所需的构象改变方面存在缺陷。我们将对突变小鼠的Ig变量和开关区进行深度测序,以确定MMR中这些突变对体细胞超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型B细胞中核心MMR蛋白的化学计量和相互作用来寻找导致这些表型的机制。为了在原代B细胞中实现这一目标,我们将使用在内源性基因座中表达TAP标记的MSH6和EXO1的小鼠进行免疫沉淀(IP)、串联亲和纯化(TAP)、western blot分析和质谱分析。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,我们在CH12F3细胞的shRNA筛选中发现了抑制CSR的染色质重塑蛋白,这些蛋白是MMR相互作用组的一部分。我们相信,剖析MSH6和MSH2的每个结构域的作用,确定MMR复合物和负责V区突变和类开关重组的染色质重塑因子的差异,将揭示容易出错的MMR如何在很大程度上局限于Ig基因,并最终揭示这些过程的调节变化如何在许多组织中导致癌症。
项目成果
期刊论文数量(0)
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MATTHEW D SCHARFF其他文献
MATTHEW D SCHARFF的其他文献
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{{ truncateString('MATTHEW D SCHARFF', 18)}}的其他基金
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:
8403693 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
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:
7758277 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
Mismatch Repair in V region mutation and isotype switch
V区突变和同种型转换中的错配修复
- 批准号:
7076248 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
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:
9132482 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
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:
9109534 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
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:
7579404 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
Mismatch Repair in V region mutation and isotype switch
V区突变和同种型转换的错配修复
- 批准号:
6925507 - 财政年份:2003
- 资助金额:
$ 3.13万 - 项目类别:
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