DNA damage produced by enediynes in the human phosphoglycerate kinase gene in vivo: esperamicin A1 as a nucleosome footprinting agent.

Abstract

We have used both conventional and a modified version of ligation-mediated polymerase chain reaction (LMPCR) to study the role of chromatin structure in the selection of DNA targets by three DNA-cleaving enediynes in whole cells. On the basis of previous studies of enediyne target selection in nucleosomes, we focused on nucleosomes present in the human X-linked phosphoglycerate kinase (PGK1) gene. Damage produced by esperamicin A1 in cells containing a transcriptionally inactive copy of the X-chromosome is reduced compared to that in naked DNA in two regions that encompass approximately 130 and approximately 150 base pairs upstream of the PGK1 gene. These sizes are consistent with nucleosome core DNA. Damage produced by esperamicin A1 in the transcriptionally active form of the gene, in which nucleosomes are not apparent, did not show such a pattern. Esperamicin C, an analogue of esperamicin A1 lacking an intercalating anthranilate moiety, and calicheamicin, both groove binders, were found to cleave DNA throughout the nucleosome core and linker. These results confirm hypotheses generated from studies in isolated chromatin and reconstituted nucleosomes and suggest that enediynes may prove useful as chromatin footprinting agents.