DNA repair and lg class switching

描述(申请人提供)：B细胞在被抗原激活后，经历抗体类型(同型)的转换，从表达IgM转变为表达Ig G、Ig A或Ig E，同时保持对同一抗原的特异性。由于同种类型决定了抗体的效应器功能，因此类别转换允许体液免疫反应对不同的感染性生物做出适应性反应。类转换是通过位于每个重链恒定(CH)区基因上游的开关(S)区序列之间的DNA重组事件发生的。这一过程与Ig可变区基因的体细胞超突变在机制上有相似之处。最近发现，激活诱导的胞苷脱氨酶通过使S区域内的DC残基脱氨而启动类开关重组，从而产生Du残基。产生的Du残基被尿嘧啶DNA糖基酶(UNG)去除，留下碱性残基，而没有UNG的小鼠和人进行CSR的能力大大降低。然而，为了启动CSR，必须将基本位点转化为单链DNA断裂。这被认为是由于AP内切酶(APE)，但这两种类人猿中的哪一种可能参与了这一过程尚不清楚。来自该组的数据支持另一种可能性，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启动DNA断裂是如何引入的。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区域的机制。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)途径的组成部分是否参与CSR，并确定它们的作用。目的2：确定碱基切除修复(BER)酶APE1或APE2是否在SU片段产生起始DNA断裂。目的3：确定BER和NER通路是否对起始DNA断裂的产生是冗余的，以及这些通路之间是否存在竞争。我们还将探索在不同的诱导条件下，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聚合酶亚单位是否参与S区域的易错修复以及这种参与是否受细胞因子的调节，来探讨突变是如何引入免疫球蛋白S区域的。

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.