Department of Immunology, University of Toronto, Toronto, Ontario, Canada.
PLoS Genet. 2019 Apr 4;15(4):e1008101. doi: 10.1371/journal.pgen.1008101. eCollection 2019 Apr.
Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5' and 3' overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5' overhangs were removed and 3' overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells.
类别转换重组 (CSR) 需要激活诱导胞嘧啶脱氨酶 (AID) 在 B 细胞的免疫球蛋白重链 (IGH) 触发 DNA 双链断裂 (DSB)。AID 依赖性 DSB 在 IGH 内的连接促进 CSR 和有效的体液免疫，但与非 IGH 染色体上的 DSB 连接会导致染色体易位。因此，需要仔细检查 AID 依赖性 DSB 的修复机制。AID 在 IGH 中的随机活性导致了一系列 DSB 结构。在本报告中，我们研究了 DSB 结构如何影响导致 CSR 和人类 B 细胞中染色体易位的末端连接，对于 CSR 模型效率低下且不易获得。我们使用 CRISPR/Cas9 在 IGH 和非 IGH 基因中模拟 AID 依赖性 DSB，发现与平头 DSB 相比，带有 5' 和 3' 突出端的 DSB 导致末端连接过程中的加工增加。我们观察到 5' 突出端被去除，3' 突出端在重组连接处被填充，这表明根据 DSB 极性需要不同的酶亚基进行修复。令人惊讶的是，虽然 Cas9 介导的转换无论 DSB 结构如何都优先利用 NHEJ，但 A-EJ 在没有 NHEJ 的情况下强烈偏爱修复平头 DSB，导致易位。我们发现 DSB 极性对 Cas9 介导的转换和易位的影响大于突出长度。最后，来自交错 DSB 的重组连接处表现出模板插入，表明在修复过程中进行迭代切除和填充。我们的结果表明，DSB 结构偏向于 NHEJ 或 A-EJ 修复，以完成导致 CSR 和易位的重组，从而有助于阐明人类 B 细胞中基因组重排的机制。
