DNA repair and lg class switching

DNA 修复和 LG 类别转换

• 批准号: 6853179
• 负责人: Janet M. Stavnezer
• 金额: $ 32.4万
• 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/6853179
• 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（由申请人提供）：被抗原激活后，B细胞发生抗体类别（同型）转换，从表达IgM转变为表达IgG、IgA或IgE，同时保持对同一抗原的特异性。由于同型决定了抗体的效应功能，类转换允许体液免疫反应对不同的感染性生物体作出适应性反应。类切换发生在位于每个重链常数（CH）区基因上游的开关(S)区序列之间的DNA重组事件。这一过程与Ig可变区基因的体细胞超突变具有机制上的相似性。最近已经清楚的是，激活诱导胞苷脱氨酶（AID）通过在S区域内的dC残基脱氨，产生dU残基来启动类开关重组（CSR）。产生的dU残基被尿嘧啶DNA糖基酶（UNG）切除，留下一个基本残基，没有UNG的小鼠和人类经历CSR的能力大大降低。然而，为了启动CSR，基本位点必须转化为单链DNA断裂。这被假设是由于AP内切酶（APE），但两种APE中哪一种可能参与尚不清楚。这组数据支持了另一种可能性，表明内切酶ERCC1/XPF可能也具有这种作用。这项拨款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。目的1：确定除ERCC1/XPF外，核苷酸切除修复（NER）通路的组分是否参与CSR并确定其作用。目的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。目的3：确定BER和NER通路对于起始DNA断裂的产生是否冗余，以及这些通路之间是否存在竞争。我们还将探讨在不同诱导条件下B细胞的活化是否会改变竞争。在Aim 4中，我们将通过确定翻译DNA聚合酶iota是否参与S区容易出错的修复，以及这种参与是否受细胞因子的调节，来探索突变是如何被引入Ig S区。