New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells.

Abstract

Sites for restriction endonuclease cleavage in double helical DNA are blocked from cleavage when the photoaffinity drug trimethylpsoralen is photobound at or near the site. In general, Hind III sites are about 15 fold more sensitive to inactivation than the other restriction sites which were tested, although sensitivity of different Hind III sites seems to vary somewhat depending on base sequences adjacent to the site. Hind III sites can be inactivated in two ways; one which completely blocks action of the specific restriction endonuclease and one permitting the introduction of a swivel which relaxes DNA supercoiling without producing a double strand break. Nucleosomes and perhaps other protein-DNA complexes can protect the underlying DNA sequence from trimethylpsoralen photobinding and thus protect restriction sites from inactivation. This property can be exploited to determine if specific sites are accessible to the psoralon probe in vivo and thus to establish if specific nucleotide sequences are nucleosome associated. Using this procedure evidence is obtained that nucleosomes on SV40 DNA in living infected cells are either distributed randomly or at many discrete alternate sites that approach a random distribution.