SINGLE-TURNOVER KINETICS OF HELICASE-CATALYZED DNA UNWINDING MONITORED CONTINUOUSLY BY FLUORESCENCE ENERGY-TRANSFER
SINGLE-TURNOVER KINETICS OF HELICASE-CATALYZED DNA UNWINDING MONITORED CONTINUOUSLY BY FLUORESCENCE ENERGY-TRANSFER
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DOI：
10.1021/bi00251a044
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发表时间：
1994-11-29
期刊：
影响因子：
2.9
通讯作者：
LOHMAN, TM
中科院分区：
文献类型：
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作者：
BJORNSON, KP;AMARATUNGA, M;LOHMAN, TM
我们描述了一种荧光测定法，可用于连续实时监测解旋酶催化的双链 DNA 解旋。该测定基于这样的观察：荧光共振能量转移 (FRET) 发生在供体（荧光素）和受体（六氯荧光素）荧光团之间，由于它们共价连接到双链寡脱氧核苷酸互补链的 3' 和 5' 端，因此它们非常接近。 FRET 导致双链 DNA 底物中荧光素的荧光发射强度相对于荧光素标记的单链 DNA 观察到的荧光发射强度降低。因此，在解旋酶催化双链 DNA 解旋和互补链分离时，荧光素增强（lambda(ex) = 492 nm；lambda(em) = 520 nm）。荧光测定极其灵敏，允许在荧光停流实验中以低至 1 nM 的 DNA 浓度连续监测 DNA 解旋反应。我们展示了在预稳态、单周转研究中使用这种 DNA 底物，研究由大肠杆菌 Rep 解旋酶催化的双链 DNA 解旋，并通过荧光停流进行监测。通过与使用快速化学猝灭流技术进行的相同动力学研究进行比较，我们表明荧光增强可以监测 Rep 催化的 DNA 解旋。在 1 nM DNA 下进行的单周转动力学研究作为过量 Rep 浓度的函数表明，包含 3'-ss-(dT)(20) 尾部的 18 碱基对双链体的 Rep 催化解旋是双相的，可以通过两个指数项之和来描述。观察到的第一相速率常数与 [Rep] (20-300 nM) 无关，并测量结合在生产复合物中的 Rep 二聚体对双链 DNA 的快速单周转解旋（1.3 +/- 0.2 s(-1)；23 +/- 3 个碱基对 s(-1)，25.0 摄氏度）。观察到的第二相速率常数随 [Rep] 线性增加，反映 DNA 解旋受到 Rep 结合事件的限制，双分子速率常数为 (1.8 +/- 0.1) x 10(5) M(-1) s(-1)，这可能反映 DNA 上 Rep 二聚化的速率常数。动力学竞争研究表明，两个 Rep 亚基都稳定地结合到生产复合物中的 DNA 底物上，该复合物在快速相中解开。这些动力学研究的结果与 Rep 催化 DNA 解旋的主动滚动机制一致 [Wong, I., & Lohman, T. M., (1992) Science 256, 350]。这种荧光测定应该极大地促进解旋酶催化 DNA 解旋的进一步机制研究。
We describe a fluorescence assay that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. The assay is based on the observation that fluorescence resonance energy transfer (FRET) occurs between donor (fluorescein) and acceptor (hexachlorofluorescein) fluorophores that are in close proximity due to their covalent attachment to the 3' and 5' ends of the complementary strands of a duplex oligodeoxynucleotide. FRET results in a reduction in the fluorescence emission intensity of fluorescein in the duplex DNA substrate relative to that observed for fluorescein-labeled single stranded DNA. Therefore, an enhancement of fluorescein (lambda(ex) = 492 nm; lambda(em) = 520 nm) occurs upon helicase-catalyzed unwinding of the duplex DNA and separation of the complementary strands. The fluorescence assay is extremely sensitive, allowing DNA unwinding reactions to be monitored continuously at DNA concentrations as low as 1 nM in a fluorescence stopped-flow experiment. We demonstrate the use of this DNA substrate in pre-steady state, single turnover studies of duplex DNA unwinding catalyzed by the Escherichia coli Rep helicase, monitored by fluorescence stopped flow. We show that the fluorescence enhancement monitors Rep-catalyzed DNA unwinding by comparisons with identical kinetic studies carried out using rapid chemical quench-flow techniques. Single turnover kinetic studies performed at 1 nM DNA as a function of excess Rep concentration show that Rep-catalyzed unwinding of an 18 base pair duplex containing a 3'-ss-(dT)(20) tail is biphasic and can be described by the sum of two exponential terms. The observed rate constant of the first phase is independent of [Rep] (20-300 nM) and measures the rapid single turnover unwinding of the duplex DNA by Rep dimers bound in productive complexes (1.3 +/- 0.2 s(-1); 23 +/- 3 base pairs s(-1) at 25.0 degrees C). The observed rate constant for the second phase increases linearly with [Rep], reflecting DNA unwinding that is limited by a Rep binding event occurring with a bimolecular rate constant of (1.8 +/- 0.1) x 10(5) M(-1) s(-1), which may reflect the rate constant for Rep dimerization on DNA. Kinetic competition studies indicate that both Rep subunits are bound stably to the DNA substrate in the productive complex that is unwound in the fast phase. The results of these kinetic studies are consistent with an active, rolling mechanism for Rep-catalyzed unwinding of DNA [Wong, I., & Lohman, T. M., (1992) Science 256, 350]. This fluorescence assay should greatly facilitate further mechanistic studies of helicase-catalyzed DNA unwinding.