Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.
Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.
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DOI：
10.1016/j.celrep.2014.02.022
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发表时间：
2014-03-27
期刊：
影响因子：
8.8
通讯作者：
Ha T
中科院分区：
文献类型：
--
作者：
Syed S;Pandey M;Patel SS;Ha T
噬菌体T7 gp 4作为复制解旋酶的模型蛋白，其将脱氧胸苷三磷酸（dTTP）水解与定向运动和DNA链分离偶联。我们采用单分子荧光共振能量转移方法来解决T7解旋酶解旋过程中的步骤。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>35%鸟嘌呤-胞嘧啶（GC）碱基对的双链体，我们在解旋期间观察到每2- 3bp的随机暂停。每次暂停的停留时间呈非指数分布，与每次解旋步骤前的两轮或三轮dTTP水解一致。此外，我们观察到在低dTTP浓度下，富含GC的DNA上的酶的向后运动。我们的数据表明碱基对解绕和dTTP水解之间的耦合比为1：1，并且它们进一步支持核酸马达可以具有不同大小的步骤的层次结构或者可以在过渡到后续阶段之前积累弹性能量的概念。单DNA解旋试验重现序列依赖性解旋高分辨率数据显示解旋步长为2-3 bp在解旋步骤之前有两个或三个隐藏的步骤，表明1：1化学偶联1：1偶联在低dNTP下保持，但解旋酶经常向后滑动Ha和同事提供了T7解旋酶以2-3 bp的离散步骤解旋DNA的直接证据。每个解旋步骤由两个或三个隐藏的步骤组成，表明解旋和dNTP水解的碱基对之间的偶联比为1：1，并表明在过渡到随后的阶段之前弹性能的积累。在低dNTP下，解旋酶也显示向后移动，可能是由于亚基协调的丧失。
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. Single DNA unwinding assay recapitulates sequence-dependent unwinding High-resolution data reveal an unwinding step size of 2–3 bp Two or three hidden steps precede the unwinding step, suggesting 1:1 chemical coupling 1:1 coupling is maintained at low dNTP, but helicase often slips backward Ha and colleagues provide direct evidence that the T7 helicase unwinds DNA in discrete steps of 2–3 bp. Each unwinding step consists of two or three hidden steps, suggesting a coupling ratio of 1:1 between base pairs unwound and dNTP hydrolyzed, and indicating accumulation of elastic energy, before transitioning to a subsequent phase. At low dNTP, the helicase also displays backward movements, likely due to loss of subunit coordination.
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影响因子：
56.9
作者：
Ali, JA;Lohman, TM
通讯作者：
Lohman, TM
影响因子：
14.9
作者：
Fili N;Mashanov GI;Toseland CP;Batters C;Wallace MI;Yeeles JT;Dillingham MS;Webb MR;Molloy JE
通讯作者：
Molloy JE
影响因子：
4.8
作者：
Ahnert, P;Patel, SS
通讯作者：
Patel, SS
影响因子：
2.4
作者：
Betterton, MD;Jülicher, F
通讯作者：
Jülicher, F
DOI：
10.1073/pnas.83.11.3746
发表时间：
1986-06-01
影响因子：
11.1
作者：
BRESLAUER, KJ;FRANK, R;MARKY, LA
通讯作者：
MARKY, LA