Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Biochem J. 2012 Nov 15;448(1):115-25. doi: 10.1042/BJ20121031.
The RAG (recombination-activating gene) complex is responsible for the generation of antigen receptor diversity by acting as a sequence-specific nuclease. Recent studies have shown that it also acts as a structure-specific nuclease. However, little is known about the factors regulating this activity at the genomic level. We show in the present study that the proximity of a V(D)J nonamer to heteroduplex DNA significantly increases RAG cleavage and binding efficiencies at physiological concentrations of MgCl(2). The position of the nonamer with respect to heteroduplex DNA was important, but not orientation. A spacer length of 18 bp between the nonamer and mismatch was optimal for RAG-mediated DNA cleavage. Mutations to the sequence of the nonamer and deletion of the nonamer-binding domain of RAG1 reinforced the role of the nonamer in the enhancement in RAG cleavage. Interestingly, partial mutation of the nonamer did not significantly reduce RAG cleavage on heteroduplex DNA, suggesting that even cryptic nonamers were sufficient to enhance RAG cleavage. More importantly, we show that the fragile region involved in chromosomal translocations associated with BCL2 (B-cell lymphoma 2) can be cleaved by RAGs following a nonamer-dependent mechanism. Hence our results from the present study suggest that a non-B DNA can replace the heptamer of RSS (recombination signal sequence) when present adjacent to nonamers, explaining the generation of certain chromosomal translocations in lymphoid malignancies.
RAG（重组激活基因）复合物通过充当序列特异性核酸酶负责产生抗原受体多样性。最近的研究表明，它也充当结构特异性核酸酶。然而，关于在基因组水平上调节这种活性的因素知之甚少。在本研究中我们表明，V(D)J 九聚体与异源双链 DNA 的接近显着增加了在生理浓度的 MgCl2 下 RAG 切割和结合效率。九聚体相对于异源双链 DNA 的位置很重要，但方向不重要。在九聚体和错配之间的间隔为 18 个碱基对是 RAG 介导的 DNA 切割的最佳条件。九聚体序列的突变和 RAG1 的非结合域的缺失增强了九聚体在增强 RAG 切割中的作用。有趣的是，九聚体的部分突变并没有显着降低异源双链 DNA 上的 RAG 切割，这表明即使是隐匿性九聚体也足以增强 RAG 切割。更重要的是，我们表明与 BCL2（B 细胞淋巴瘤 2）相关的染色体易位涉及的脆弱区域可以通过 RAG 沿着依赖九聚体的机制进行切割。因此，我们本研究的结果表明，当存在于九聚体附近时，非 B DNA 可以替代 RSS（重组信号序列）的七聚体，解释了淋巴恶性肿瘤中某些染色体易位的产生。
