DNA Breaks in Class Switch Recombination

描述（由申请人提供）：本提案旨在检测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IgM恒定区基因被删除并被下游恒定区基因（如C?）取代的重组事件。这种重组发生在被称为开关(S)区域的每个恒定区域基因上游的DNA中。由于中间的DNA作为一个圆圈被切除，这个过程需要在上游和下游的S区都产生双链断裂。DNA断裂会导致突变、易位和肿瘤，因此必须严格调控。众所周知，激活诱导胞苷脱氨酶（AID）在这一过程中是必需的，它可以使DNA中的胞苷脱氨，从而产生可致突变和/或导致DNA断裂的尿嘧啶。

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.