UV-laser crosslinking of proteins to DNA.
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.
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Centre de Recherche en Cancérologie et Departement de Biochimie, Faculté de Médecine, l'Université Laval, Hp5otel-Dieu de Quebec, Canada.
Dimitrov
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Houde
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D000256:Adenoviridae / Q000235:genetics; D017504:Adenovirus E4 Proteins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D007834:Lasers; D011401:Promoter Regions, Genetic; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014466:Ultraviolet Rays
D017504:Adenovirus E4 Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D004247:DNA