Mutual Inhibition of RecQ Molecules in DNA Unwinding
Mutual Inhibition of RecQ Molecules in DNA Unwinding
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DNA解旋中RecQ分子的相互抑制
DOI：
10.1074/jbc.m110.104299
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发表时间：
2010-05-21
影响因子：
4.8
通讯作者：
Xi, Xu Guang
中科院分区：
文献类型：
--
作者：
Pan, Bing-Yi;Dou, Shuo-Xing;Xi, Xu Guang
解旋酶通过水解NTP使双链体DNA（或RNA）解旋而进行构象变化和机械运动。大多数解旋酶需要单链突出端以加载到双链体DNA底物上。已经观察到一些解旋酶随着单链DNA尾长度的增加而表现出增强的解旋效率，这是通过防止解旋DNA的再退火和通过补偿前导单体的过早解离来实现的。在这里，我们报告了一个以前未知的相互抑制相邻单体的DNA解旋的单体大肠杆菌RecQ解旋酶。与单分子荧光共振能量转移显微镜，我们观察到，在饱和浓度的RecQ的解旋起始更延迟的长尾DNA，而不是短尾DNA。在单次和多次周转条件下的停流动力学研究中，对于长尾底物，解旋效率随着酶浓度的增加而降低。此外，观察到RecQ和DNA在5 '-腺苷酰-β，γ-亚氨二磷酸存在下的预孵育减轻了抑制。我们提出，相互抑制作用的结果，从一个领先的单体的两个RecA样结构域之间的裂缝的强制关闭的尾随之一，因此，两个单体的前向运动被阻止ATP结合到领先的。这种效应代表了RecQ的两个RecA样结构域在DNA解旋中相对运动的直接证据。它可能发生在所有超家族I和II解旋酶拥有两个RecA样结构域。
Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains.