AID in somatic mutation of immunoglobulin genes

AID 免疫球蛋白基因体细胞突变

• 批准号: 7573127
• 负责人: URSULA B STORB
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7573127
• 关键词: Affect; Affinity; Antibodies; Autoimmunity; B-Lymphocytes; Base Excision Repairs; Biological Process; Cancer Etiology; Cell Cycle; Cell Line; Cells; Chickens; Complex; Cytidine; Cytidine Deaminase; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Deamination; Disease; Exhibits; Frequencies; G1 Phase; Gene Conversion; Genes; Genetic Transcription; Histones; Immune response; Immunoglobulin Constant Region; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; Lymphocyte; Malignant Neoplasms; Methods; Mismatch Repair; Mitosis; Modeling; Molecular; Mus; Mutate; Mutation; Parents; Pattern; Phase; Poly A; Polymerase; Positioning Attribute; Process; Recruitment Activity; Research; Role; Single-Stranded DNA; Site; Somatic Mutation; Superhelical DNA; System; Testing; Time; Transcript; Travel; Uridine; Wild Type Mouse; antigen binding; base; gene terminator; insertion/deletion mutation; interest; novel; novel strategies; pathogen; promoter; public health relevance; repaired; research study; response; tool; transcription termination; transition mutation

DESCRIPTION (provided by applicant): The process of somatic hypermutation (SHM) of immunoglobulin genes is initiated by the cytidine deaminase AID creating cytidine (C) to uridine (U) transition mutations. Outside of SHM, U is faithfully repaired to C with the help of base excision repair (BER) and mismatch repair (MMR). During SHM, paradoxically, these repair mechanisms are recruited together with translesion DNA polymerases to create mutations at and near the U by error-prone repair. A critical hallmark of SHM is the restriction of the mutations to the 5' end of Ig genes, including the V(D)J region, but sparing the constant region. In this way, an enormous diversity of antigen-binding variable regions is created while protecting the biological functions of the antibody constant regions. We have considered two ways in which this V-region restriction may come about. First, AID may only be active in the 5' region, and second, AID may act throughout the gene but error-prone repair would only operate in the 5' region. In extensive experiments with mice that are defective in BER or MMR or both, we found the same pattern of mutations as in wild type mice, namely restriction to the variable region and its immediate flanks. This leads to the conclusion that AID does not act at the 3' portion of the Ig gene. We now wish to determine how exactly AID interacts with the sequences in which it deaminates Cs. Based on experiments where the constant region was mutated when the Ig gene promoter was duplicated in front of the constant region, we proposed the following model: AID associates with the transcription complex near the promoter, travels with the RNA polymerase during transcript elongation, deaminates Cs in single- stranded DNA arising in negative DNA supercoils behind the polymerase, and dissociates from the transcription complex within ~2kbp from the promoter due to limited affinity. We propose a novel and untried approach to test this model. The experiments will be carried out with a modified chicken B cell line, DT40 PseudoV-del, that suffers cytydine deaminations and subsequent SHM at a high rate. The modified line was produced by J.M. Buerstedde by deleting the pseudo V genes of the parent DT40 line so that the modified cells cannot carry out Ig gene conversion. In response to AID, the modified pseudoV-del DT40 cells undergo only SHM and do so at a high rate since AID- induced uridines are not directed toward gene conversion. They are therefore an ideal tool to study SHM. If the results of these exploratory experiments are clear, either supporting or rejecting the model of SHM described above, they will provide a platform and methods to unravel remaining questions, such as how AID is targeted only to Ig and some other genes, including some of known relevance to cancer and other diseases. PUBLIC HEALTH RELEVANCE: This is a proposal to study the hypermutation of antibody genes. This process is beneficial, because it can result in highly specific and efficient antibodies against pathogens and cancer. However, it is also dangerous, because it can cause cancer of lymphocytes and autoimmunity.

描述（由申请人提供）：免疫球蛋白基因的体细胞超突变（SHM）的过程是由胞苷脱氨酶辅助引起的，从而产生胞苷（C）到尿苷（U）过渡突变。在SHM之外，U在基本切除修复（BER）和不匹配维修（MMR）的帮助下忠实地修复了C。在SHM期间，自相矛盾的是，这些修复机制与跨DNA聚合酶一起募集，以通过易于易错的修复在U在U处和附近产生突变。 SHM的关键标志是将突变限制为Ig基因的5'末端，包括V（d）J区域，但会保留恒定区域。这样，在保护抗体常数区域的生物学功能的同时，创建了抗原结合变量区域的巨大多样性。我们考虑了这种V区限制可能产生的两种方式。首先，援助只能在5'区域中活跃，其次，援助可以在整个基因中起作用，但容易出错的修复只能在5'区域内运行。在用BER或MMR或MMR有缺陷的小鼠进行的广泛实验中，我们发现了与野生型小鼠相同的突变模式，即限制到可变区域及其直接的侧面。这得出的结论是，艾滋病不在Ig基因的3'部分起作用。现在，我们希望确定AID如何与其脱离CS的序列相互作用。基于在恒定区域前重复Ig基因启动子在恒定区域重复时突变恒定区域的实验，我们提出了以下模型：与启动子附近的转录复合物相关，与启动子附近的转录复合物相关，与RNA聚合酶在转录过程中的RNA聚合酶在单链DNA中脱离了DNA的cs，在单链DNA中脱离了DNA comprients conmenters and dna compriections conflessise and Polymiatise and Polymasase，＆由于亲和力有限，来自发起人。我们提出了一种新颖而未经尝试的方法来测试该模型。实验将使用改良的鸡B细胞系DT40 Pseudov-del进行，该系列会遭受cytydine脱氨酸的影响，然后以高速率进行SHM。修改线是由J.M. Buerstedde生成的，它通过删除父dt40系的伪V基因，从而使修饰的细胞无法执行Ig基因转换。为了响应AID，改良的Pseudov-Del DT40细胞仅经历SHM，并且以高速率进行，因为辅助诱导的尿苷不是针对基因转化的。因此，它们是研究SHM的理想工具。如果这些探索性实验的结果是明确的，则支持或拒绝上述SHM模型，它们将提供一种平台和方法来解开其余问题，例如AID仅针对IG和其他一些基因，包括与癌症和其他疾病的一些已知相关性。公共卫生相关性：这是研究抗体基因超数的建议。该过程是有益的，因为它可以导致针对病原体和癌症的高度特异性和有效的抗体。但是，这也很危险，因为它会导致淋巴细胞和自身免疫性癌。