Tutkus Marijonas, Sasnauskas Giedrius, Rutkauskas Danielis
Institute of Physics, Center for Physical Sciences and Technology, Savanoriu 231, Vilnius, 02300, Lithuania.
Institute of Biotechnology, Vilnius University, Sauletekio av. 7, Vilnius, 10257, Lithuania.
Biopolymers. 2017 Dec;107(12). doi: 10.1002/bip.23075. Epub 2017 Oct 27.
Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.
许多II型限制性内切核酸酶需要两个识别序列拷贝以实现最佳活性。此类酶同时结合两个DNA位点会产生一个DNA环。在此，我们利用表面固定的DNA片段的单分子荧光共振能量转移（smFRET）来研究四聚体内切核酸酶NgoMIV诱导的DNA环化动力学。我们使用了一个带有两个NgoMIV识别位点的DNA片段和一对FRET染料，使得在蛋白质诱导的DNA环化过程中，染料彼此靠近，从而产生FRET信号。DNA与NgoMIV相互作用的动力学证明是异质的，各个smFRET轨迹表现出广泛不同的平均环化状态持续时间。不同类型的动力学归因于不同类型的DNA - 蛋白质复合物，其由一个同时结合到两个特定位点的NgoMIV四聚体介导（“慢”轨迹），或者由两个结合DNA的NgoMIV四聚体的半特异性相互作用介导（“快”轨迹），以及单个NgoMIV分子的构象异质性。
