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.

中文翻译：

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UbaLAI是IIE型单体限制性酶

II型限制性核酸内切酶（REases）形成了一大批高度多样化的酶。即使是对共同识别位点具有特异性的REase，其寡聚结构，域结构和DNA切割机制也常常有所不同。在这里，我们报告了单体限制性核酸内切酶UbaLAI的生化和结构表征，该酶对假对称DNA序列5'-CC / WGG-3'具有特异性（其中W = A / T，'/'表示切割位置）。我们提出了UbaLAI N末端结构域（UbaLAI-N）的1.6Å共晶体结构，并显示它类似于5'-CCWGG-3'序列特异的EcoRII的B3家族结构域。我们还发现，UbaLAI C末端结构域（UbaLAI-C）与单体REase MvaI密切相关，后者是5'-CCWGG-3'序列特有的另一种酶。UbaLAI的动力学研究表明，它需要两个识别位点才能发挥最佳活性，并且像其他类型的IIE酶一样，它使用一个识别位点的副本刺激第二个副本的切割。我们建议在反应过程中，UbaLAI-N充当将单体UbaLAI-C域束缚到DNA的句柄，从而帮助UbaLAI-C在单个DNA结合事件中在第二个识别位点上执行两个连续的DNA切口反应。类似的反应机制可能是其他单体两结构域REase的特征。从而帮助UbaLAI-C在单个DNA结合过程中在第二个识别位点上进行两个连续的DNA切口反应。类似的反应机制可能是其他单体两结构域REase的特征。从而帮助UbaLAI-C在单个DNA结合过程中在第二个识别位点上进行两个连续的DNA切口反应。类似的反应机制可能是其他单体两结构域REase的特征。