Lu Catherine P, Sandoval Hector, Brandt Vicky L, Rice Phoebe A, Roth David B
Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, and Department of Pathology, New York University School of Medicine, New York, New York 10016, USA.
Nat Struct Mol Biol. 2006 Nov;13(11):1010-5. doi: 10.1038/nsmb1154. Epub 2006 Oct 8.
The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.
Rag蛋白通过一种机制上类似于剪切粘贴转座的过程来进行V(D)J重组。具体而言，Rag复合物利用Rag1中催化性的天冬氨酸-天冬氨酸-谷氨酸（DDE）三联体，通过直接酯交换反应形成DNA发夹结构。如何引入足够的DNA扭曲以允许发夹结构形成呢？我们推测，与某些转座酶一样，Rag蛋白可能利用芳香族氨基酸残基来稳定一个翻转出来的碱基。通过体内和体外实验以及结构预测，我们确定了Rag1中对发夹结构形成至关重要的残基。其中一个保守的色氨酸（Trp893）可能参与切割位点附近的碱基堆积相互作用，分别与Tn5、Tn10和Hermes转座酶中的Trp298、Trp265和Trp319类似。催化谷氨酸周围的其他残基（YKEFRK）可能与IS4家族转座酶中的YREK基序具有功能相似性。
