Transcription-induced Mutations

转录诱导的突变

• 批准号: 7459855
• 负责人: ASHOK S BHAGWAT
• 金额: $ 25.73万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7459855
• 关键词: 8-oxoguanine; Affinity; Antibodies; Antigens; Bacteria; Biochemical Genetics; Biological Assay; Class; Cytosine; DNA; DNA Repair Enzymes; DNA repair protein; DNA-Directed DNA Polymerase; Deamination; Dependence; Escherichia coli; Genes; Genetic; Genetic Transcription; Guanine; Human; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; In Vitro; Individual; Induced Mutation; Infection; Invaded; Kanamycin Resistance; Length; Mammalian Cell; Mammalian Genetics; Modeling; Mutagenesis; Mutation; Mutation Spectra; Phleomycins; Process; Proteins; Resistance; Role; Role playing therapy; Score; Shapes; Site; Structure; Syndrome; System; Testing; Thymine; Time; Uracil; Virus; activation-induced cytidine deaminase; bacterial genetics; base; fight against; human AICDA protein; research study; response; role model; stem

DESCRIPTION (provided by applicant): Antibody maturation occurs in response to invasion of a foreign agent such as a virus and creates antibody proteins that have greater affinity towards the invading antigens. Activation-induced cytidine deaminase (AID) is required for this process and its essential role in the fight against infections is evidenced by the fact that individuals that are unable to make active AID suffer from hyper-IgM syndrome. An important feature of the role of AID in promoting somatic hypermutations (SHM) and class switch recombination -2 processes essential for antibody maturation - is the requirement for the transcription of the immunoglobulin gene. We propose here to investigate the transcription-dependence of SHM. We outline here experiments in vitro, in Escherichia coli and in mammalian cells that will investigate the role of AID in promoting cytosine to thymine (C to T) and other base substitutions. Specifically, we will investigate the effects of supercoiling, R-loop formation and other structural features on strand bias and clustering of mutations. The experiments will use genetic assays in which kanamycin-resistant and phleomycin-resistant revertants respectively score C to T and non-C-to-T mutations. We will also develop a mammalian genetic system to analyze these 2 classes of mutations and study their strand bias during AID promoted hypermutations. Further, this system will be used to study the roles played by various DNA repair enzymes, DNA polymerases and accessory proteins in shaping the strand bias and spectrum of mutations responsible for antibody maturation.

描述（由申请人提供）：抗体成熟是响应病毒等外来因子的入侵而发生的，并产生对入侵抗原具有更大亲和力的抗体蛋白。该过程需要激活诱导的胞苷脱氨酶 (AID)，无法产生活性 AID 的个体患有高 IgM 综合征，这一事实证明了它在对抗感染中的重要作用。 AID 在促进体细胞超突变 (SHM) 和抗体成熟所必需的类别转换重组 -2 过程中的作用的一个重要特征是免疫球蛋白基因转录的需要。我们在此建议研究 SHM 的转录依赖性。我们在此概述了在大肠杆菌和哺乳动物细胞中进行的体外实验，这些实验将研究 AID 在促进胞嘧啶至胸腺嘧啶（C 至 T）和其他碱基取代中的作用。具体来说，我们将研究超螺旋、R 环形成和其他结构特征对链偏向和突变聚类的影响。实验将使用基因检测，其中卡那霉素抗性和腐草霉素抗性回复体分别对C到T和非C到T突变进行评分。我们还将开发一个哺乳动物遗传系统来分析这两类突变并研究它们在 AID 促进的超突变期间的链偏向。此外，该系统将用于研究各种 DNA 修复酶、DNA 聚合酶和辅助蛋白在形成链偏向和负责抗体成熟的突变谱中所发挥的作用。