UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides.
An efficient method of cross-linking DNA to protein is described. The method is based on the incorporation of photoactive 8-azidoadenine 2'-deoxyribonucleotides into DNA. We have found that 8-N3dATP is a substrate for E. coli DNA polymerase I and that 8-N3dATP can be incorporated into plasmid pBR322 by nick-translation. Subsequently we were able to cross-link a set of different proteins to 8-azido-2'-deoxyadenosine-containing pBR322 by UV irradiation (366nm). No DNA-protein photocross-linking was observed under the same conditions when the non-photoactive plasmid pBR322 was used.
D003432:Cross-Linking Reagents; D004256:DNA Polymerase I / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis; D004926:Escherichia coli / Q000201:enzymology; D006657:Histones / Q000378:metabolism*; D010957:Plasmids / Q000528:radiation effects*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D003838:Deoxyadenine Nucleotides; D006657:Histones; C057680:8-azido-2'-deoxyadenosine-5'-triphosphate; D004256:DNA Polymerase I