Role of ABH1's AP endonuclease activity in immunoglobulin gene diversification

描述(申请人提供)：ABH1‘S AP内切酶活性在免疫球蛋白基因多样化中的作用：这项建议的重点是确定ABH1(大肠杆菌AlkB DNA修复酶的哺乳动物同源物I)是否在淋巴细胞中切割基本位点并产生断裂，这是类开关重组(CSR)和基因转换(GC)所必需的中间产物，并可能促进B细胞亲和力成熟过程中特定类型的体细胞超突变(SH)。这些过程是由激活诱导的胞苷脱氨酶(AID)作用于V区或切换区的单链DNA而启动的。尿嘧啶N-糖基酶(Ung)作用于脱氨基胞苷(现为尿嘧啶)，产生碱性位点。我们最近对ABH1的生化研究表明，它具有内切核活性，对双链DNA中的碱基位置具有特异性切割。ABH1与CSR、GC或SHM之间没有直接联系的报道，但没有证据确凿地表明这些过程中存在任何已知的基本位点内切酶。如果ABH1是负责切割AID/UNG诱导的B淋巴细胞发育中的基本位点的酶，那么这项研究将对理解B细胞分化的几个关键步骤产生很大影响，从而大大推动该领域的进展。本研究的具体目标是：1.从ABH1+/FLOX小鼠培育ABH1-/-小鼠。2.确定ABH1-/-小鼠B细胞CSR是否正常进行。3.确定ABH1-/-小鼠B细胞SHM是否发生改变。公共卫生相关性：高等脊椎动物在正常环境中的生存需要高度适应性的免疫系统来产生大量不同的抗原受体。特别是，激活诱导脱氨酶(AID)和尿嘧啶DNA N-糖基酶(Ung)在DNA中创建基本的位点，这是类切换重组、基因转换和体细胞超突变所必需的步骤。这项建议中描述的研究将确定ABH1，即大肠杆菌AlkB DNA修复酶的人类同源物，是否利用其新检测到的能力切割DNA的基本位置，以促进B细胞中免疫球蛋白基因的多样化系统。

DESCRIPTION (provided by applicant): Role of ABH1's AP endonuclease activity in immunoglobulin gene diversification: The focus of this proposal is to determine whether ABH1 (mammalian homologue I of the Escherichia coli AlkB DNA repair enzyme) functions in lymphocytes to cleave abasic sites and generate breaks that are the requisite intermediate for class switch recombination (CSR) and gene conversion (GC) and that may promote certain types of somatic hypermutations (SH) during B cell affinity maturation. These processes are known to be initiated by activation induced cytidine deaminase (AID) acting on single-stranded DNA in V or switch regions. Uracil N-glycosylase (Ung) acts on the deaminated cytidine (now a uracil) to generate an abasic site. Our recent biochemical studies of ABH1 revealed it to possess an endonucleolytic activity with specificity for cleaving at abasic sites in double-stranded DNA. No direct connection has been reported between ABH1 and CSR, GC, or SHM, but no evidence firmly implicating any known abasic site endonuclease in these processes has been forthcoming. If ABH1 is the enzyme responsible for cleaving AID/Ung induced abasic sites in developing B lymphocytes, this study will have high impact for understanding several critical steps in B cell differentiation, substantially advancing progress in the field. The specific goals of the proposed research are to: 1. Create ABH1-/- mice from ABH1+/flox mice. 2. Determine whether CSR proceeds normally in B cells from ABH1-/- mice. 3. Determine whether SHM is altered in B cells from ABH1-/- mice. PUBLIC HEALTH RELEVANCE: Survival of higher vertebrates in normal environments requires a highly adaptive immune system to generate an immense repertoire of distinct antigen receptors. In particular, activation-induced deaminase (AID) and uracil DNA N-glycosylase (Ung) create abasic sites in DNA as required steps of class switch recombination, gene conversion, and somatic hypermutation. The studies described in this proposal will identify whether ABH1, the human homologue of the Escherichia coli AlkB DNA repair enzyme, uses its newly-detected ability to cleave abasic sites of DNA to facilitate immunoglobulin gene diversification system in B cells.