Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.
Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics.
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DOI：
10.1093/nar/gkt1297
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发表时间：
2014-03
影响因子：
14.9
通讯作者：
Bianco P
中科院分区：
文献类型：
--
作者：
Bongini L;Melli L;Lombardi V;Bianco P
在DNA65pN的张力下，双链∼经历了从基本(B-型)构象到1.7倍长的构象的过度拉伸转变，其性质直到最近才开始被理解。在这里，我们通过记录施加在不同熔化度和温度(10-25°C)的λ噬菌体DNA上的力步骤的长度瞬变来提供这一转变的结构和热力学特征。超拉伸力下降20-35pN后的缩短瞬变显示出一系列双链延伸(S形式)片段的快速缩短和由于熔化片段的重新退火而暂停的现象。在2-35pN拉伸到超拉伸力后的拉长瞬变显示出两态反应动力学，并表明整个70%的拉伸是先于熔化且独立于熔化的B-S相变。伸长瞬变的温度依赖性表明，对B-S转变的熵贡献是热熔化熵变的三分之一，这加强了双链S形式保持相当一部分链间键的证据。单位伸长率(22bp)的协同性与温度无关，表明结构因素，如核酸序列，控制着转变。
Under a tension of ∼65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10–25°C). The shortening transient following a 20–35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2–35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition.
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