A Preparative Method for Crosslinking Proteins to DNA in Nuclei by Single-pulse UV Laser Irradiation

Abstract— Photocrosslinking of proteins to DNA by single-pulse UV laser has been used only in analytical experiments, carried out with reconstituted complexes of a single DNA binding protein and a labeled target sequence. Here we propose a large-scale technique for irradiation of nuclei, generating preparative quantities of covalently linked protein-DNA complexes for further analysis of the partner molecules. The use of a flow cuvette allows a milligram of DNA in either nuclei or chromatin to be irradiated by a single pulse within few minutes. The efficiency of crosslinking varies from 6 to 12% of the total nuclear proteins. The presence of histones and other chromosomal proteins in the crosslinked protein-DNA complexes was demonstrated by using specific antibodies. The irradiation procedure can be fully automated using a microcomputer.