Kriatchko Aleksei N, Anderson Dirk K, Swanson Patrick C
Dept. of Medical Microbiology and Immunology, Creighton University Medical Center, 2500 California Plaza, Omaha, NE 68178, USA.
Mol Cell Biol. 2006 Jun;26(12):4712-28. doi: 10.1128/MCB.02487-05.
RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis.
RAG-1和RAG-2通过在重组信号序列处切割DNA启动V(D)J重组，该过程通过连续的切口和转酯反应产生平端信号末端和以DNA发夹结构终止的编码末端。普遍存在的DNA修复因子随后介导断裂DNA的重新连接。V(D)J重组遵循12/23规则，该规则将重排限制在RAG蛋白结合的保守七聚体和九聚体序列之间具有不同长度DNA（12或23个碱基对）的信号序列上。两种RAG蛋白都经历了广泛的诱变，揭示了一个或两个切割步骤所需的残基或参与DNA末端连接过程的残基。功能获得性RAG突变体仍未被鉴定。在这里，我们报告了一种新的RAG-1突变体E649A，它通过优先增强发夹形成在体外支持更高的切割活性。RAG-1突变对DNA结合活性和其他DNA链转移反应（如转座）的催化作用没有实质性影响。然而，与野生型对应物不同，12/23调节的联会并不能强烈刺激含有E649A RAG-1的RAG复合物的切割活性。有趣的是，在细胞培养中，野生型和E649A RAG-1对含有12/23对信号序列的质粒底物的切割和重组水平相似；然而，在12/12质粒底物上，E649A RAG-1支持的切割和重组比野生型RAG-1多约三倍。这些数据表明，E649A RAG-1突变可能会干扰RAG蛋白感知12/23调节联会的能力。
