PicoNewton-Millisecond Force Steps Reveal the Transition Kinetics and Mechanism of the Double-Stranded DNA Elongation
PicoNewton-Millisecond Force Steps Reveal the Transition Kinetics and Mechanism of the Double-Stranded DNA Elongation
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DOI：
10.1016/j.bpj.2011.06.039
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发表时间：
2011-08-17
影响因子：
3.4
通讯作者：
Lombardi, Vincenzo
中科院分区：
文献类型：
--
作者：
Bianco, Pasquale;Bongini, Lorenzo;Lombardi, Vincenzo
利用双激光光镊在25 ℃的力钳条件下研究了噬菌体双链DNA从基本构象（B态）到1.7倍长的部分解绕构象（S态）的过度拉伸转变动力学。我们的压电伺服系统具有前所未有的分辨率，可以施加0.5-2 pN的毫秒级力阶跃，揭示了伸长动力学的指数特征，并允许我们测试B-S转变机制的两态性质。通过分析拉伸速率常数的负载依赖性，我们发现基本的拉伸步长为5.85 nm，表明类似于25个碱基对的协同效应。这种机制将基元反应的自由能增加到类似于94 k（B）T，解释了DNA基本构象的稳定性，并解释了为什么ds-DNA可以在过度拉伸时保持平衡。
We study the kinetics of the overstretching transition in lambda-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25 degrees C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5-2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of similar to 25 basepairs. This mechanism increases the free energy for the elementary reaction to similar to 94 k(B)T, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.