Activation‐induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of immunoglobulin genes in B cells, whereas off‐targeted AID activity contributes to oncogenic mutations and chromosomal translocations associated with B cell malignancies. Paradoxically, only a minority of AID is allowed to access the nuclear genome, but the majority of AID is retained in the cytoplasm. It is unknown whether cytoplasmic AID can access and target the mitochondrial genome [mitochondrial DNA (mtDNA)]. To address this issue, we developed high‐fidelity differential DNA denaturation PCR, which allowed the enrichment of genuine mtDNA mutations and therefore the identification of endogenous mtDNA mutation signatures in vitro. With this approach, we showed that AID targeting to mtDNA is a rare event in AID‐expressing lymphoma lines. Further biochemical and microscopic analysis revealed that a fraction of cytosol AID is associated with the outer membrane of mitochondria but unable to access the mitochondrial matrix. Together, our data suggested that the mitochondrial genome is protected from AID‐mediated mutagenesis by physical segregation of AID from accessing mtDNA within the mitochondrial matrix.

中文翻译：

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优化的高保真 3DPCR 以评估活化诱导的胞苷脱氨酶的潜在线粒体靶向。

激活诱导的胞苷脱氨酶 (AID) 启动 B 细胞中免疫球蛋白基因的体细胞超突变和类别转换重组，而脱靶 AID 活性有助于与 B 细胞恶性肿瘤相关的致癌突变和染色体易位。矛盾的是，只允许少数 AID 进入核基因组，但大部分 AID 保留在细胞质中。尚不清楚细胞质 AID 是否可以访问并靶向线粒体基因组 [线粒体 DNA (mtDNA)]。为了解决这个问题，我们开发了高保真差异 DNA 变性 PCR，它允许富集真正的 mtDNA 突变，从而在体外鉴定内源性 mtDNA 突变特征. 通过这种方法，我们发现 AID 靶向 mtDNA 在表达 AID 的淋巴瘤细胞系中是罕见的事件。进一步的生化和显微镜分析表明，一小部分细胞质 AID 与线粒体外膜相关，但无法进入线粒体基质。总之，我们的数据表明，通过将 AID 与线粒体基质内的 mtDNA 进行物理隔离，可以保护线粒体基因组免受 AID 介导的诱变。