Syed Salman, Pandey Manjula, Patel Smita S, Ha Taekjip
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Cell Rep. 2014 Mar 27;6(6):1037-1045. doi: 10.1016/j.celrep.2014.02.022. Epub 2014 Mar 13.
Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2-3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase.
噬菌体T7 gp4作为复制解旋酶的模型蛋白，它将三磷酸脱氧胸苷（dTTP）水解与定向移动及DNA链分离偶联起来。我们采用单分子荧光共振能量转移方法来解析T7解旋酶解开DNA过程中的各个步骤。我们证实，T7解旋酶的解旋速率随着碱基对稳定性的增加而降低。对于含有超过35%鸟嘌呤-胞嘧啶（GC）碱基对的双链体，我们观察到在解旋过程中每2 - 3个碱基对就会出现随机停顿。每个停顿处的停留时间呈非指数分布，这与每个解旋步骤之前进行两到三轮dTTP水解一致。此外，我们在低dTTP浓度下观察到该酶在富含GC的DNA上出现向后移动。我们的数据表明解开的碱基对与dTTP水解之间的偶联比为1:1，并且进一步支持了核酸马达可以有不同大小步骤的层级结构，或者在过渡到后续阶段之前可以积累弹性能量这一概念。
