Blanco J, Kimura H, Mueller G C
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Anal Biochem. 1987 Jun;163(2):537-45. doi: 10.1016/0003-2697(87)90260-0.
Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [alpha-32P] dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins--marked by short covalently linked radioactive DNA tags--are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.
本文描述了两种在HeLa细胞核DNA复制位点鉴定DNA-蛋白质相互作用的方法。在此过程中，新复制的DNA链首先分别通过掺入[α-32P]dCTP和溴脱氧尿苷三磷酸在体外进行标记和光敏化。然后用紫外光照射，使与新复制DNA的光敏化和同位素标记链相邻的蛋白质发生共价交联。在用核酸酶消化大部分DNA后，通过短共价连接的放射性DNA标签标记的交联蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶中进行电泳分离，并通过放射自显影进行检测。利用这项技术，已证明某些蛋白质能选择性地与新复制的DNA结合。该方法似乎适用于解决各种涉及DNA-蛋白质结合的问题。
