Bastianello Giulia, Arakawa Hiroshi
IFOM - FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy.
Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, 20133 Milan, Italy.
Nucleic Acids Res. 2017 Jan 9;45(1):231-243. doi: 10.1093/nar/gkw887. Epub 2016 Oct 3.
All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries.
免疫球蛋白（Ig）基因重塑系统的所有三种B细胞特异性活性——基因转换、体细胞超突变和类别转换重组——都需要激活诱导脱氨酶（AID）。AID诱导的DNA损伤必须进一步加工并分解为不同的DNA重组途径。为了表征Ig基因转换的潜在中间体，我们在AID敲除DT40细胞系的Ig轻链基因座的互补决定区1（CDR1）中插入了一个I-SceI识别位点，并条件性表达I-SceI内切酶。在此，我们表明，在没有AID的情况下，CDR1中的双链断裂（DSB）足以触发Ig基因转换。DSB诱导的基因转换的模式和假基因使用情况与AID诱导的基因转换相当；令人惊讶的是，有时单个DSB会诱导多个基因转换事件。这些构成了直接证据，表明V区的DSB可以是基因转换的中间体。DSB下游的DNA损伤的命运比AID的情况具有更大的灵活性，这提示了两种替代模型：（i）与单链断裂（SSB）相比，生理基因转换过程中的DSB占少数，SSB在DNA脱氨后经常产生，或者（ii）生理基因转换由严格调控的DSB介导，该DSB在局部受到保护，免受非同源末端连接（NHEJ）或其他非同源DNA重组机制的影响。
