DETECTION OF PROTEIN - DNA INTERACTIONS AT BETA-GLOBIN GENE-CLUSTER IN INTACT HUMAN-CELLS UTILIZING HEDAMYCIN AS DNA-DAMAGING AGENT

The DNA sequence specificity of hedamycin (HDM) damage was investigate d in the single-copy human beta-globin gene cluster in an erythroid ce ll line, a nonerytihroid cell line, and purified genomic DNA. The targ et DNA sequences for this study were the beta-globin gene locus contro l region (LCR) hypersensitive site 2 (HS-2) and the beta-globin gene p romoter. The DNA fragments produced by HDM damage in these target sequ ences were selectively amplified by the ligation-mediated polymerase c hain reaction (LMPCR) and analyzed at nucleotide resolution by DNA-seq uencing gel electrophoresis. The DNA sequences damaged by HDM in the c ellular environment were found to be similar to that observed in the p urified genomic DNA. However, substantial differences did occur betwee n the intensity of cellular and purified genomic DNA reaction products at discrete regions corresponding to transcription factor-blinding mo tifs. This was most apparent in the LCR HS-2 at the tandem NF-E2/AP-1 motif, where the DNA damage activity of HDM was severely impaired. Thi s motif has been shown to bind to the erythroid-specific nuclear facto r-erythroid 2 (NF-E2) and the widely distributed activator protein-1 ( AP-1). The HDM damage protection patterns or ''genomic footprints'' ob served at this motif were probably caused by protein-DNA interactions with one or both of these transcription factors. This result indicates that the DNA damaging activity of HDM in cells is sensitive to bound nuclear factors. Because HDM can enter intact cells, where its DNA dam aging activity is modulated by protein-DNA interactions, it may have a pplication in genomic footprinting experiments.