UV-LASER CROSS-LINKING OF PROTEINS TO DNA

Photochemical crosslinking is now a powerful method for studying prote in-nucleic acid interactions. UV light is a zero-length crosslinking a gent that predominantly or exclusively crosslinks proteins to nucleic acids at their contact points. It can therefore provide strong evidenc e 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. Su ch 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 cross linking may be delivered in time intervals on the order of nano or eve n picoseconds. We described detailed procedures for UV laser-induced p rotein-DNA crosslinking both in vivo and in vitro. Technical aspects, including the choice of UV laser for irradiation, the isolation of cov alently crosslinked protein-DNA complexes, immunochemical techniques f or both the identification and isolation of specific protein-DNA compl exes and the identification of the crosslinked DNA sequences, are revi ewed in detail. The application of UV laser crosslinking in kinetic st udies is illustrated by the example of the TATA-binding protein (TBP) interaction with the adenovirus E4 promoter. (C) 1997 Academic Press.