High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein-DNA complexes.
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.
Castella
Sandrine
S
Institute for Cancer Studies, University of Sheffield, Beech Hill Rd., Sheffield S10 2RX, UK.
Sanders
Cyril M
CM
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000528:radiation effects; D015342:DNA Probes / Q000138:chemical synthesis / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry; D008969:Molecular Sequence Data; D010777:Photochemistry; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D015342:DNA Probes; D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda