Castella Sandrine, Sanders Cyril M
Institute for Cancer Studies, University of Sheffield, Beech Hill Rd., Sheffield S10 2RX, UK.
Anal Biochem. 2006 Dec 15;359(2):203-9. doi: 10.1016/j.ab.2006.09.018. Epub 2006 Oct 12.
Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2-ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5'-phosphorylated strand of a polymerase chain reaction product with lambda exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [alpha-(32)P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein-DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2-ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein-DNA complexes.
光化学交联已被广泛用于鉴定与DNA上特定位点相互作用的蛋白质。结合蛋白的鉴定通常依赖于通过交联将放射性标记从DNA转移到蛋白质上。我们着手对一个由与DNA结合的两个蛋白二聚体组成的小型病毒复制起始前复合物——牛乳头瘤病毒E1E2-ori复合物进行精细定位。在此，我们描述了一种简单的方法，用于生成具有紧邻标记核苷酸的苯基叠氮光活化交联基团的高比活性探针。该方法基于用λ外切核酸酶选择性破坏聚合酶链反应产物的一条5'-磷酸化链，并用硫代磷酸酯取代的寡核苷酸、[α-(32)P]dNTP和嗜热酶重建探针。我们还开发了一种高分辨率凝胶内交联测定法来探测特定的蛋白质-DNA复合物。通过这些方法，我们获得了乳头瘤病毒E1E2-ori起始前复合物的结构信息，否则这些信息很难获得。这些方法应广泛适用于蛋白质-DNA复合物的研究。
