Sasnauskas Giedrius, Tamulaitiene Giedre, Tamulaitis Gintautas, Calyševa Jelena, Laime Migle, Rimšeliene Renata, Lubys Arvydas, Siksnys Virginijus
Institute of Biotechnology, Vilnius University, Sauletekio av. 7, LT-10257 Vilnius, Lithuania.
Thermo Fisher Scientific Baltics, V. A. Graiciuno str. 8, LT-02241, Vilnius, Lithuania.
Nucleic Acids Res. 2017 Sep 19;45(16):9583-9594. doi: 10.1093/nar/gkx634.
Type II restriction endonucleases (REases) form a large and highly diverse group of enzymes. Even REases specific for a common recognition site often vary in their oligomeric structure, domain organization and DNA cleavage mechanisms. Here we report biochemical and structural characterization of the monomeric restriction endonuclease UbaLAI, specific for the pseudosymmetric DNA sequence 5'-CC/WGG-3' (where W = A/T, and '/' marks the cleavage position). We present a 1.6 Å co-crystal structure of UbaLAI N-terminal domain (UbaLAI-N) and show that it resembles the B3-family domain of EcoRII specific for the 5'-CCWGG-3' sequence. We also find that UbaLAI C-terminal domain (UbaLAI-C) is closely related to the monomeric REase MvaI, another enzyme specific for the 5'-CCWGG-3' sequence. Kinetic studies of UbaLAI revealed that it requires two recognition sites for optimal activity, and, like other type IIE enzymes, uses one copy of a recognition site to stimulate cleavage of a second copy. We propose that during the reaction UbaLAI-N acts as a handle that tethers the monomeric UbaLAI-C domain to the DNA, thereby helping UbaLAI-C to perform two sequential DNA nicking reactions on the second recognition site during a single DNA-binding event. A similar reaction mechanism may be characteristic to other monomeric two-domain REases.
II型限制性内切核酸酶（REases）构成了一个庞大且高度多样化的酶类群。即使是对共同识别位点具有特异性的REases，其寡聚结构、结构域组织和DNA切割机制也常常有所不同。在此，我们报告了单体限制性内切核酸酶UbaLAI的生化和结构特征，该酶对假对称DNA序列5'-CC/WGG-3'具有特异性（其中W = A/T，且'/'表示切割位置）。我们展示了UbaLAI N端结构域（UbaLAI-N）的1.6 Å共晶体结构，并表明它类似于对5'-CCWGG-3'序列具有特异性的EcoRII的B3家族结构域。我们还发现UbaLAI C端结构域（UbaLAI-C）与单体REase MvaI密切相关，MvaI是另一种对5'-CCWGG-3'序列具有特异性的酶。对UbaLAI的动力学研究表明，它需要两个识别位点才能达到最佳活性，并且与其他IIE型酶一样，利用一个识别位点的拷贝来刺激另一个拷贝的切割。我们提出，在反应过程中，UbaLAI-N充当一个柄，将单体UbaLAI-C结构域与DNA拴系在一起，从而帮助UbaLAI-C在单个DNA结合事件期间对第二个识别位点进行两次连续的DNA切口反应。类似的反应机制可能是其他单体双结构域REases的特征。
