Moss T, Dimitrov S I, Houde D
Centre de Recherche en Cancérologie et Departement de Biochimie, Faculté de Médecine, l'Université Laval, Hp5otel-Dieu de Quebec, Canada.
Methods. 1997 Feb;11(2):225-34. doi: 10.1006/meth.1996.0409.
Photochemical crosslinking is now a powerful method for studying protein-nucleic acid interactions. UV light is a zero-length crosslinking agent that predominantly or exclusively crosslinks proteins to nucleic acids at their contact points. It can therefore provide strong evidence for close protein-nucleic acid interactions. However, to achieve an acceptable degree of crosslinking with conventional UV light sources, exposure times ranging from minutes to several hours are necessary. Such prolonged irradiation allows for the artifactual redistribution of proteins and precludes kinetic studies. The use of UV lasers overcomes these difficulties since the number of photons required for the crosslinking may be delivered in time intervals on the order of nano- or even picoseconds. We described detailed procedures for UV laser-induced protein-DNA crosslinking both in vivo and in vitro. Technical aspects, including the choice of UV laser for irradiation, the isolation of covalently crosslinked protein-DNA complexes, immunochemical techniques for both the identification and isolation of specific protein-DNA complexes and the identification of the crosslinked DNA sequences, are reviewed in detail. The application of UV laser crosslinking in kinetic studies is illustrated by the example of the TATA-binding protein (TBP) interaction with the adenovirus E4 promoter.
光化学交联现已成为研究蛋白质-核酸相互作用的一种强大方法。紫外线是一种零长度交联剂，主要或仅在蛋白质与核酸的接触点将它们交联在一起。因此，它可为紧密的蛋白质-核酸相互作用提供有力证据。然而，要使用传统紫外光源实现可接受的交联程度，曝光时间需要从几分钟到几个小时不等。这种长时间照射会导致蛋白质出现人为重新分布，从而无法进行动力学研究。使用紫外激光克服了这些困难，因为交联所需的光子数可以在纳秒甚至皮秒量级的时间间隔内传递。我们描述了体内和体外紫外激光诱导蛋白质-DNA交联的详细程序。详细综述了技术方面的内容，包括用于照射的紫外激光的选择、共价交联的蛋白质-DNA复合物的分离、用于鉴定和分离特定蛋白质-DNA复合物以及鉴定交联DNA序列的免疫化学技术。以TATA结合蛋白（TBP）与腺病毒E4启动子的相互作用为例，说明了紫外激光交联在动力学研究中的应用。
