Abstract

The photoactivatable modified oligonucleotides were used to investigate direct contacts formed by the type IIE EcoRII restriction endonuclease and the T/A bases of its recognition site (5'-CC^T/_AGG). EcoRII dimer consists of a central catalytic core, made of two C-terminal endonuclease-like domains (EcoRII-C) from different subunits, and two N-terminal effector DNA binding domains (EcoRII-N). According to co-crystal structure of isolated EcoRII-C with DNA catalytic dimer EcoRII-C flips nucleotides of the central T/A pair into the enzyme binding pockets. Нere, photocross-linking technique was used to investigate the direct contacts formed by extrahelical T/A bases in the protein pockets of full-length EcoRII within the pre-reactive EcoRII–DNA complex obtained in the presence of Ca²⁺ in solution. Photoreactive zero-length agent 5-iodo-2'-deoxyuridine (IdU) was introduced as single substituent into the central T/A position of EcoRII recognition site or into the flanking nucleotide sequences of 14-mer DNA substrate. The substitution of only dT or dA residues in EcoRII recognition site resulted in formation of photocross-links upon irradiation only in the presence of Ca²⁺. Proteolytic digestion of the enzyme-oligonucleotide conjugates followed by MALDI-MS analysis have allowed to identify the ²²⁴VEYD²²⁷ EcoRII region involved in the formation of the cross-links. This region belongs to the central part of H-10 α-helix. Y²²⁶ residue was suggested to form cross-link with T or A bases of EcoRII site replaced by IdU within the pre-reactive complex. The flipped base pair protein pockets of EcoRII seem to accommodate equally well both A and T bases of the DNA substrate. Altogether, IdU-containing photoactivatable DNA substrates have allowed to trap the flipped bases in complex with full-length EcoRII before DNA cleavage in the solution and to identify direct enzyme–DNA contacts important for high specificity of EcoRII for the Т/A nucleotides providing a highly specific cleavage reaction.

中文翻译：

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EcoRII限制性核酸内切酶与其目标序列的碱基形成特异性接触，该目标序列从具有光活化底物的过渡复合物中的DNA跃迁而来。

摘要

使用可光活化的修饰寡核苷酸来研究IIE EcoRII型限制性核酸内切酶及其识别位点的T / A碱基（5'-CC ^T / _A GG）形成的直接接触。EcoRII二聚体由一个中央催化核心组成，该核心由来自不同亚基的两个C末端核酸内切酶样结构域（EcoRII-C）和两个N末端效应子DNA结合结构域（EcoRII-N）组成。根据分离的EcoRII-C与DNA催化二聚体的共晶体结构，EcoRII-C将中央T / A对的核苷酸翻转到酶结合口袋中。使用光交联技术，研究了存在Ca时获得的预反应EcoRII-DNA复合物中全长EcoRII蛋白质口袋中螺旋外T / A碱基形成的直接接触。解决方案中的²⁺。将光反应性零长度试剂5-碘-2'-脱氧尿苷（IdU）作为单个取代基引入EcoRII识别位点的中心T / A位置或14-mer DNA底物的侧翼核苷酸序列中。仅在Ca ²⁺存在下，照射后EcoRII识别位点中仅dT或dA残基的取代导致形成光交联。酶-寡核苷酸共轭物的蛋白水解消化，然后进行MALDI-MS分析，已经鉴定出参与交联形成的²²⁴ VEYD ²²⁷ EcoRII区。该区域属于H-10α-螺旋的中心部分。ÿ ²²⁶建议该残基与在反应前复合物中的IdR取代的EcoRII位点的T或A碱基形成交联。EcoRII的碱基对翻转后的蛋白质口袋似乎可以很好地容纳DNA底物的A和T碱基。总的来说，含有IdU的可光活化DNA底物允许在溶液中进行DNA切割之前，将与完整EcoRII结合的翻转碱基捕获，并鉴定对于EcoRII对Т/ A核苷酸具有高特异性的重要酶-DNA直接接触。高特异性裂解反应。