DNA Breaks in Class Switch Recombination

描述(由申请人提供)：这项建议是为了检查在免疫球蛋白类开关重组(CSR)过程中发生的DNA断裂。CSR是B淋巴细胞交换其产生的免疫球蛋白(Ig)分子的恒定区域以最有效地对抗它们所暴露的病原体的过程。CSR涉及一种重组事件，即IgM恒定区基因缺失，并被下游恒定区基因如C？取代。这种重组发生在每个恒定区基因上游的DNA中，称为开关(S)区。由于中间的脱氧核糖核酸被切割成一个环，这个过程需要在S的上游和下游区域都进行双链断裂。DNA断裂可能导致突变、易位和肿瘤，因此必须受到严格监管。众所周知，激活诱导胞苷脱氨酶(AID)是这一过程所必需的，它可以使DNA中的胞苷脱氨基产生尿嘧啶，从而产生突变和/或导致DNA断裂的尿嘧啶。 这里提出的实验将确定艾滋病是否直接作用于体内免疫球蛋白S区域的脱氧核糖核酸，将胞苷转化为尿嘧啶，并将确定导致断裂形成的后续步骤。连接介导的聚合酶链式反应将被用来检测S区域DNA的确切断裂点，并检验这一假说，即由艾滋病病毒活性引发的断裂最初是单链和交错双链断裂，但DNA修复酶的末端处理可以将其中一些断裂转化为钝化的双链断裂。将在错配修复(MMR)、核苷酸切除修复(NER)和碱基切除修复(BER)途径中缺失DNA修复蛋白的小鼠细胞中检查断裂的位置和结构。BER酶UNG和APE可以从DNA中去除尿嘧啶，并切割DNA骨架，被认为参与了断裂的形成。来自BER途径的酶将用于处理诱导转换的B细胞的基因组DNA，以检测该模型预测的修复途径中的中间产物。

DESCRIPTION (provided by applicant): This proposal is to examine breaks in DNA that occur during the process of immunoglobulin class switch recombination (CSR). CSR is the process by which B lymphocytes exchange the constant region of the immunoglobulin (Ig) molecule they produce in order to most effectively combat the pathogen to which they have been exposed. CSR involves a recombination event in which the IgM constant region gene is deleted and replaced by a downstream constant region gene such as C?. The recombination occurs within DNA located upstream of each constant region gene known as switch (S) regions. As the intervening DNA is excised as a circle, the process requires that double strand breaks are made in both the upstream and downstream S regions. DNA breaks can lead to mutations, translocations and tumors and therefore must be tightly regulated. It is known that activation-induced cytidine deaminase (AID) is required for this process and that it can deaminate cytidines in DNA to generate uracils that can be mutagenic and/or lead to DNA breaks. The experiments proposed here will determine if AID acts directly on Ig S region DNA in vivo to convert cytidines to uracils and also will determine the subsequent steps that lead to break formation. Ligation-mediated (LM)-PCR will be used to detect the exact breakpoint in S region DNA and to test the hypothesis that the breaks instigated by AID activity are initially single-stranded and staggered double strand breaks (DSBs), but that end-processing by DNA repair enzymes can convert some of these breaks to blunt DSBs. The position and structure of breaks will be examined in cells from mice deficient in DNA repair proteins from the mismatch repair (MMR), nucleotide excision repair (NER), and base excision repair (BER) pathways. The BER enzymes UNG and APE can remove uracil from DNA and nick the DNA backbone and are thought to be involved in break formation. Enzymes from the BER pathway will be used to treat genomic DNA from B cells induced to switch in order to detect intermediates in the repair pathway that are predicted by this model.