DNA-DAMAGE PRODUCED BY ENEDIYNES IN THE HUMAN PHOSPHOGLYCERATE KINASEGENE IN-VIVO - ESPERAMICIN A1 AS A NUCLEOSOME FOOTPRINTING AGENT

We have used both conventional and a modified version of ligation-medi ated polymerase chain reaction (LMPCR) to study the role of chromatin structure in the selection of DNA targets by three DNA- cleaving enedi ynes in whole cells, On the basis of previous studies of enediyne targ et selection in nucleosomes, we focused on nucleosomes present in the human X-linked phosphoglycerate kinase (PGK1) gene. Damage produced by esperamicin Al in cells containing a transcriptionally inactive copy of the X-chromosome is reduced compared to that in naked DNA in two re gions that encompass similar to 130 and similar to 150 base pairs upst ream of the PGK1 gene. These sizes are consistent with nucleosome core DNA, Damage produced by esperamicin Al in the transcriptionally activ e form of the gene, in which nucleosomes are not apparent, did not sho w such a pastern. Esperamicin C, an analogue of esperamicin Al lacking an intercalating anthranilate moiety, and calicheamicin, both groove binders, were found to cleave DNA throughout the nucleosome core and l inker, These results confirm hypotheses generated from studies in isol ated chromatin and reconstituted nucleosomes and suggest that enediyne s may prove useful as chromatin footprinting agents.