DNA Breaks in Class Switch Recombination

类别转换重组中的 DNA 断裂

• 批准号: 7062491
• 负责人: CAROL E SCHRADER
• 金额: $ 27.73万
• 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: 2005-05-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062491
• 关键词: B lymphocyte; DNA damage; DNA repair; aminohydrolases; cytidine; enzyme mechanism; gene rearrangement; immunoglobulin genes; laboratory mouse; nucleic acid structure; uracil

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.

描述（由申请方提供）：本提案旨在检查免疫球蛋白类别转换重组（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）分子的恒定区以最有效地对抗其所暴露的病原体的过程。CSR涉及IgM恒定区基因缺失并被下游恒定区基因（如C？）取代的重组事件。重组发生在位于每个恒定区基因上游的DNA内，称为开关（S）区。由于插入的DNA作为环状被切除，该过程需要在上游和下游S区中进行双链断裂。DNA断裂可能导致突变、易位和肿瘤，因此必须严格调控。已知该过程需要活化诱导的胞苷脱氨酶（AID），并且其可使DNA中的胞苷脱氨基以产生尿嘧啶，尿嘧啶可具有诱变性和/或导致DNA断裂。 本文提出的实验将确定AID是否在体内直接作用于IG S区DNA以将胞苷转化为尿嘧啶，并且还将确定导致断裂形成的后续步骤。连接介导的（LM）-PCR将用于检测S区DNA中的确切断裂点，并检验以下假设：AID活性引发的断裂最初是单链和交错双链断裂（DSB），但DNA修复酶的末端加工可将其中一些断裂转化为钝DSB。将在来自DNA修复蛋白缺陷小鼠的细胞中检查断裂的位置和结构，所述DNA修复蛋白来自错配修复（MMR）、核苷酸切除修复（NER）和碱基切除修复（BER）途径。BER酶UNG和APE可以从DNA中去除尿嘧啶并使DNA骨架产生切口，并且被认为参与断裂形成。来自BER途径的酶将用于处理来自诱导转换的B细胞的基因组DNA，以检测由该模型预测的修复途径中的中间体。