DNA-binding orientation and domain conformation of the E-coli Rep helicase monomer bound to a partial duplex junction:: Single-molecule studies of fluorescently labeled enzymes
DNA-binding orientation and domain conformation of the E-coli Rep helicase monomer bound to a partial duplex junction:: Single-molecule studies of fluorescently labeled enzymes
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DOI：
10.1016/j.jmb.2003.12.031
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发表时间：
2004-02-13
影响因子：
5.6
通讯作者：
Ha, T
中科院分区：
文献类型：
--
作者：
Rasnik, I;Myong, S;Ha, T
SF 1 DNA解旋酶是一种多结构域蛋白，可在与ATP结合和水解偶联的反应中解链双链体DNA。两个这样的解旋酶，大肠杆菌Rep和嗜热脂肪芽孢杆菌PcrA的晶体结构表明，这些蛋白质的2B亚结构域可以发现在显着不同的方向（封闭与开放）相对于其余的蛋白质，这表明2B结构域是高度灵活的。通过在单分子水平上系统地利用荧光共振能量转移，我们确定了E。coliRep单体在溶液中与3 '-单链-双链（ss/ds）DNA连接处结合，以及其2B亚结构域的相对取向。为了实现这一目标，我们开发了一种高效的程序，用于位点特异性荧光标记的Rep和生物友好的固定化方案，保留其活动。进行了合奏和单分子实验，虽然单分子实验被证明是必不可少的，在这里提供定量的距离信息，不能通过稳态合奏测量。使用距离约束的三角测量程序，我们表明，在解决方案中的2B亚结构域的Rep单体主要是在“封闭”的构象时，结合到一个3 '-SS/DS DNA，类似于在复合物中观察到的方向PcrA结合到一个3'-SS/DS DNA。以前的生物化学研究表明，与这种3 '-ss/ds DNA底物结合的Rep单体不能解旋DNA，并且Rep寡聚体是解旋酶活性所必需的。因此，与部分双链体DNA结合的Rep的闭合形式似乎是酶的抑制形式。(C)2004 Elsevier Ltd.保留所有权利。
The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E. coli Rep monomer bound to a 3'-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the "closed" conformation when bound to a 3'-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3'-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3'-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme. (C) 2004 Elsevier Ltd. All rights reserved.