The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway
The run-on oligomer filament enzyme mechanism of SgrAI: Part 2. Kinetic modeling of the full DNA cleavage pathway
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DOI：
10.1074/jbc.ra118.003682
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发表时间：
2018-07
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影响因子：
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通讯作者：
Chad K. Park;Jonathan L. Sanchez;Claudia J. Barahona;L. Basantes;Juan A. Sanchez;Christian Hernandez;N. Horton
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文献类型：
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作者：
Chad K. Park;Jonathan L. Sanchez;Claudia J. Barahona;L. Basantes;Juan A. Sanchez;Christian Hernandez;N. Horton
由酶形成的纤维或低聚物现在被认为是一种广泛存在的现象，具有潜在独特的酶调节特性和生物学作用。SgrAI是一种变构型II型限制性内切酶，可形成具有激活DNA切割活性和改变DNA序列特异性的低聚细丝。在这个由两部分组成的工作中，我们测量了运行中的低聚物长丝机制中的各个步骤，以解决协同性，捕获，长丝生长机制和活性封存的具体问题，使用荧光团标记的DNA，动态FRET测量和反应建模与全局数据拟合。最终模型和速率常数表明，在低至中等浓度的SgrAI-DNA下，SgrAI-DNA复合物与低聚物丝结合的组装步骤相对缓慢（比扩散限制慢3-4个数量级），并且具有速率限制。在运行的低聚物丝中，分解SgrAI - DNA复合物是下一个最慢的步骤，但其速度足以限制任何一个SgrAI或DNA拷贝在动态丝中的停留时间。此外，DNA的切割速率常数在连接的低聚物丝中比在分离的SgrAI-DNA复合物中快4个数量级，并且比SgrAI-DNA复合物从连接的低聚物丝中解离要快，这使得反应效率很高，因为每次与丝的结合都可能在丝解离之前导致DNA的切割。
Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI–DNA complexes into the run-on oligomer filament is relatively slow (3–4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI–DNA. The disassembly step involving dissociation of complexes of SgrAI–DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI–DNA complexes and faster than dissociation of SgrAI–DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.