DNA Breaks in Class Switch Recombination

类别转换重组中的 DNA 断裂

• 批准号: 7172602
• 负责人: CAROL E SCHRADER
• 金额: $ 26.96万
• 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/7172602
• 关键词: APEX1 gene; Aftercare; B-Lymphocytes; Base Excision Repairs; Biological Assay; Cells; Characteristics; Cytidine; DNA; DNA Repair Enzymes; DNA Repair Pathway; DNA Single Strand Break; DNA Structure; DNA repair protein; DNA-(apurinic or apyrimidinic site) lyase; Data; Deamination; Deoxyribose; Enzymes; Event; Excision; Frequencies; Genetic; Genetic Recombination; Genomics; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin M; Immunoglobulin Switch Recombination; Immunoglobulins; In Vitro; Lead; Ligation; Lyase; Mediating; Methods; Mismatch Repair; Modeling; Molecular; Mus; Mutation; Nucleotide Excision Repair; Pathway interactions; Polymerase Chain Reaction; Positioning Attribute; Process; Proteins; Site; Specificity; Structure; Testing; Thinking; Time; Uracil; Vertebral column; activation-induced cytidine deaminase; base; constant region gene; endonuclease; in vivo; pathogen; repair enzyme; repaired; research study; tumor; uracil-DNA glycosylase

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（由申请人提供）：该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) 分子恒定区的过程，以便最有效地对抗它们所接触的病原体。 CSR涉及重组事件，其中IgM恒定区基因被删除并被下游恒定区基因例如Cα取代。重组发生在位于每个恒定区基因（称为开关 (S) 区）上游的 DNA 内。由于插入的 DNA 被切除为环状，因此该过程需要在上游和下游 S 区域均产生双链断裂。 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，该过程需要激活诱导的胞苷脱氨酶 (AID)，它可以使 DNA 中的胞苷脱氨基，生成尿嘧啶，尿嘧啶可能具有诱变性和/或导致 DNA 断裂。 这里提出的实验将确定 AID 是否在体内直接作用于 Ig S 区 DNA，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 (LM)-PCR 将用于检测 S 区 DNA 中的确切断裂点，并检验 AID 活性引发的断裂最初是单链和交错双链断裂 (DSB) 的假设，但 DNA 修复酶的末端处理可以将其中一些断裂转化为平端 DSB。将在缺乏来自错配修复（MMR）、核苷酸切除修复（NER）和碱基切除修复（BER）途径的DNA修复蛋白的小鼠的细胞中检查断裂的位置和结构。 BER 酶 UNG 和 APE 可以从 DNA 中去除尿嘧啶并在 DNA 主链上产生切口，并且被认为参与断裂形成。 BER 途径中的酶将用于处理诱导转换的 B 细胞的基因组 DNA，以便检测该模型预测的修复途径中的中间体。