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

描述（申请人提供）：ABH 1的AP核酸内切酶活性在免疫球蛋白基因多样化中的作用：本提案的重点是确定ABH 1是否（大肠杆菌AlkB DNA修复酶的哺乳动物同源物I）在淋巴细胞中起作用以切割脱碱基位点并产生断裂，所述断裂是类别转换重组（CSR）和基因转换（GC）的必要中间体。并且在B细胞亲和力成熟过程中可能促进某些类型的体细胞超突变（SH）。已知这些过程是通过激活诱导的胞苷脱氨酶（AID）作用于V或开关区域中的单链DNA而启动的。尿嘧啶N-糖基化酶（Ung）作用于脱氨基的胞苷（现为尿嘧啶）以产生脱碱基位点。我们最近对ABH 1的生物化学研究表明，它具有内切核酸酶活性，对双链DNA中的脱碱基位点具有特异性切割。目前还没有报道ABH 1与CSR、GC或SHM之间存在直接联系，但没有证据表明这些过程中存在任何已知的无碱基位点核酸内切酶。如果ABH 1是负责切割发育中的B淋巴细胞中AID/Ung诱导的脱碱基位点的酶，则该研究将对理解B细胞分化中的几个关键步骤具有高度影响，从而实质性地推进该领域的进展。本研究的具体目标是：1.从ABH 1 +/flox小鼠创建ABH 1-/-小鼠。2.确定CSR是否在ABH 1-/-小鼠的B细胞中正常进行。3.确定ABH 1-/-小鼠的B细胞中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。特别地，活化诱导的脱氨酶（AID）和尿嘧啶DNA N-糖基化酶（Ung）在DNA中产生脱碱基位点，作为类别转换重组、基因转换和体细胞超突变的必要步骤。本提案中描述的研究将确定大肠杆菌Alk B DNA修复酶的人类同源物ABH 1是否使用其新检测到的切割DNA脱碱基位点的能力来促进B细胞中的免疫球蛋白基因多样化系统。

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.