Transcription-induced Mutations

描述（由申请人提供）：抗体成熟响应于外来因子（如病毒）的入侵而发生，并产生对入侵抗原具有更大亲和力的抗体蛋白。活化诱导的胞苷脱氨酶（AID）是这一过程所必需的，并且其在对抗感染中的重要作用由以下事实证明：不能使活性AID的个体患有高IgM综合征。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过程中的作用的一个重要特征是需要免疫球蛋白基因的转录。我们建议在这里调查SHM的转录依赖性。我们在这里概述了体外实验，在大肠杆菌和哺乳动物细胞中，将调查AID在促进胞嘧啶胸腺嘧啶（C到T）和其他碱基取代的作用。具体来说，我们将研究超螺旋，R环的形成和其他结构特征对链偏置和突变聚类的影响。这些实验将使用遗传分析，其中卡那霉素抗性和腐草霉素抗性回复突变体分别对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促进的超突变过程中的链偏好。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差和负责抗体成熟的突变谱中所起的作用。

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.