Transcription-induced Mutations

描述(由申请人提供)：抗体成熟是对外来物质如病毒的入侵作出反应，并产生对入侵抗原有更大亲和力的抗体蛋白。激活诱导胞苷脱氨酶(AID)在这一过程中是必需的，其在对抗感染中的重要作用被无法使AID激活的人患上高IgM综合征的事实所证明。AID在促进体细胞超突变(SHM)和类切换重组-抗体成熟所必需的2个过程中的作用的一个重要特征是对免疫球蛋白基因的转录的要求。我们建议在这里研究SHM的转录依赖性。我们在这里概述了在体外，在大肠杆菌和哺乳动物细胞中的实验，这些实验将研究AID在促进胞嘧啶到胸腺嘧啶(C到T)和其他碱基替换中的作用。具体地说，我们将研究超螺旋、R环形成和其他结构特征对突变的链偏向和聚集性的影响。这些实验将使用基因分析，其中卡那霉素耐药和苯丙霉素耐药突变株分别对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向。此外，该系统将被用来研究各种DNA修复酶、DNA聚合酶和辅助蛋白在形成导致抗体成熟的突变的链偏向和谱方面所起的作用。

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.