Asynchrony of Base-Pair Breaking and Nucleotide Releasing of Helicases in DNA Unwinding
Asynchrony of Base-Pair Breaking and Nucleotide Releasing of Helicases in DNA Unwinding
复制标题
DNA 解旋中碱基对断裂和解旋酶核苷酸释放的异步性
DOI：
10.1021/acs.jpcb.8b01470
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发表时间：
2018
影响因子：
3.3
通讯作者：
Lu Ying
中科院分区：
文献类型：
--
作者：
Ma Jian Bing;Jia Qi;Xu Chun Hua;Li Jing Hua;Huang Xing Yuan;Ma Dong Fei;Li Ming;Xi Xu Guang;Lu Ying
解旋酶利用核苷酸三磷酸水解的能量以离散步骤解旋双链DNA（dsDNA）。尽管深入的研究，步进的机制仍然知之甚少。在这里，我们应用单分子荧光共振能量转移来表征两个非环解旋酶，大肠杆菌RecQ（E。coliRecQ）和SaccharomycesparaePif1（ScPif1）。我们的数据表明，当具有游离突出端的叉状dsDNA用作底物时，coliRecQ和ScPif1以分布在宽范围内的不均匀步骤展开dsDNA。当张力施加在悬突上时，ScPif1的步长分布的整体轮廓变窄，而E. coliRecQ保持不变。此外，两个解旋酶的测量步长集中在半个碱基对的整数倍。我们提出了一个通用的步进机制，其中解旋酶打破一个碱基对的时间和隔离新生的核苷酸，然后释放后，随机数量的碱基对打破事件。该机制可以定量地解释所观察到的解旋模式，并提供了解旋酶活性的一般视图。
Helicases harness the energy of nucleotide triphosphate hydrolysis to unwind double-stranded DNA (dsDNA) in discrete steps. In spite of intensive studies, the mechanism of stepping is still poorly understood. Here, we applied single-molecule fluorescent resonant energy transfer to characterize the stepping of two nonring helicases,Escherichia coliRecQ (E. coliRecQ) andSaccharomyces cerevisiaePif1 (ScPif1). Our data showed that when forked dsDNA with free overhangs are used as substrates, bothE. coliRecQ and ScPif1 unwind the dsDNA in nonuniform steps that distribute over broad ranges. When tension is exerted on the overhangs, the overall profile of the step-size distribution of ScPif1 is narrowed, whereas that ofE. coliRecQ remains unchanged. Moreover, the measured step sizes of the both helicases concentrate on integral multiples of a half base pair. We propose a universal stepping mechanism, in which a helicase breaks one base pair at a time and sequesters the nascent nucleotides and then releases them after a random number of base-pair breaking events. The mechanism can interpret the observed unwinding patterns quantitatively and provides a general view of the helicase activity.