A PREPARATIVE METHOD FOR CROSS-LINKING PROTEINS TO DNA IN NUCLEI BY SINGLE-PULSE UV LASER IRRADIATION

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