Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
J Biol Chem. 2010 Mar 5;285(10):7587-97. doi: 10.1074/jbc.M109.089631. Epub 2010 Jan 5.
The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C((d))C((S))C((S))" (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability.
重组激活基因 (RAG) 复合物在 V(D)J 重组过程中的序列特异性已经得到了很好的研究。RAG 还可以作为结构特异性核酸内切酶；然而，其作用机制知之甚少。在这里，我们表明，除了 DNA 结构外，序列还决定了 RAG 在改变的 DNA 结构上切割的模式和效率。当存在于异源双链 DNA 的单链区域时，胞嘧啶核苷酸优先被 RAG 切割。尽管未配对的胸腺嘧啶核苷酸也被切割，但效率要弱得多。RAG 诱导单链或双链断裂取决于胞嘧啶的位置以及它是否存在于一条或两条链上。有趣的是，当腺嘌呤或鸟嘌呤核苷酸存在于单链区域时，RAG 无法诱导断裂。紧邻泡序列的核苷酸也可能影响 RAG 切割。因此，我们提出了“C((d))C((S))C((S))”（d，双链；s，单链）作为 RAG 在单链/双链 DNA 转换处诱导断裂的共识序列。这样的共识序列基序可用于解释文献中描述的其他类型 DNA 结构上的 RAG 切割。因此，这里描述的 RAG 切割机制可以解释导致基因组不稳定性的特定于淋巴组织的染色体重排的各个方面。
