DNA repair and lg class switching

DNA 修复和 LG 类别转换

• 批准号: 7012289
• 负责人: Janet M. Stavnezer
• 金额: $ 31.7万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012289
• 关键词: B lymphocyte; DNA directed DNA polymerase; DNA repair; aminohydrolases; cytokine; endonuclease; flow cytometry; gene rearrangement; immunogenetics; immunoglobulin genes; laboratory mouse; phosphoester ligase

DESCRIPTION (provided by applicant): Upon activation by antigen, B cells undergo antibody class (isotype) switching, changing from expression of IgM to expression of IgG, IgA or IgE, while maintaining specificity for the same antigen. Since the isotype determines the effector function of the antibody, class switching allows the humoral immune response to adaptively respond to different infectious organisms. Class switching occurs by a DNA recombination event between switch (S) region sequences located upstream of each heavy chain constant (CH) region gene. This process has mechanistic similarities to somatic hypermutation of Ig variable region genes. It has recently become clear that activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by deamination of dC residues within S regions, creating dU residues. The resulting dU residues are excised by uracil DNA glycosylase (UNG) leaving a basic residues, and mice and humans without UNG have greatly reduced abilities to undergo CSR. However, in order to initiate CSR, the abasic site must be converted to a single strand DNA break. This has been hypothesized to be due to AP endonuclease (APE) but which of the two APEs might be involved are unknown. Another possibility is supported by data from this group, suggesting that the endonuclease ERCC1/XPF might also have this role. One of the goals of this grant is to determine how the initiating DNA breaks are introduced. A related goal is to determine the mechanisms for introduction of mutations into S regions during the DNA repair processes accompanying CSR. There are 4 specific aims directed towards these goals. Aim 1: to determine if components of the nucleotide excision repair (NER) pathway, in addition to ERCC1/XPF, are involved in CSR and to determine their role. Aim 2: to determine if the base excision repair (BER) enzymes APE1 or APE2 create the initiating DNA breaks in the Su segments. Aim 3: to determine if the BER and NER pathways are redundant for creation of the initiating DNA breaks and if there is a competition between these pathways. We will also explore if the competition is altered by activation of B cells in the presence of different induction conditions. In Aim 4 we will explore how the mutations are introduced into Ig S regions by determining if the translesion DNA polymerase iota is involved in the error-prone repair of S regions and if this involvement is regulated by cytokines.

描述（申请人提供）：被抗原激活后，B细胞经历抗体类别（同种型）转换，从表达IgM变为表达IgG、IgA或IgE，同时保持对相同抗原的特异性。由于同种型决定了抗体的效应子功能，类别转换使得体液免疫反应能够适应性地响应不同的感染性生物体。类别转换是通过位于每个重链恒定 (CH) 区基因上游的转换 (S) 区序列之间的 DNA 重组事件而发生的。该过程与 Ig 可变区基因的体细胞超突变具有机械相似性。最近已经清楚，激活诱导的胞苷脱氨酶 (AID) 通过 S 区域内的 dC 残基脱氨基，产生 dU 残基来启动类别转换重组 (CSR)。由此产生的 dU 残基被尿嘧啶 DNA 糖基化酶 (UNG) 切除，留下碱性残基，没有 UNG 的小鼠和人类进行 CSR 的能力大大降低。然而，为了启动 CSR，脱碱基位点必须转换为单链 DNA 断裂。据推测，这是由 AP 核酸内切酶 (APE) 引起的，但尚不清楚这两种 APE 中的哪一种可能参与其中。该小组的数据支持另一种可能性，表明核酸内切酶 ERCC1/XPF 也可能具有此作用。这笔资助的目标之一是确定如何引入起始 DNA 断裂。一个相关的目标是确定在伴随 CSR 的 DNA 修复过程中将突变引入 S 区的机制。针对这些目标有 4 个具体目标。目标 1：确定除 ERCC1/XPF 之外，核苷酸切除修复 (NER) 通路的组成部分是否参与 CSR 并确定它们的作用。目标 2：确定碱基切除修复 (BER) 酶 APE1 或 APE2 是否在 Su 片段中产生起始 DNA 断裂。目标 3：确定 BER 和 NER 途径对于产生起始 DNA 断裂是否是多余的，以及这些途径之间是否存在竞争。我们还将探讨在不同诱导条件下 B 细胞的激活是否会改变竞争。在目标 4 中，我们将通过确定跨损伤 DNA 聚合酶 iota 是否参与 S 区的易错修复以及这种参与是否受到细胞因子的调节，探讨如何将突变引入 Ig S 区。