INFLUENCE OF CHROMATIN STRUCTURE ON BLEOMYCIN-DNA INTERACTIONS AT BASE-PAIR RESOLUTION IN THE HUMAN BETA-GLOBIN GENE-CLUSTER

The DNA sequence specificity of bleomycin was examined in human cells and in purified genomic DNA. In each case, DNA damage sites were deter mined at nucleotide resolution in the human single-copy beta-globin pr omoter and the locus control region (LCR) hypersensitive site 2 (HS-2) . Exponential amplification of gene-specific genomic fragments was ach ieved by ligation-mediated PCR, and labeled reaction products were ana lyzed directly by sequencing gel electrophoresis. Bleomycin was found to cleave DNA preferentially at GC, GT, and GA dinucleotides. This stu dy represents the first occasion that the sequence specificity of bleo mycin has been determined in intact human cells at the single-copy gen e level. The intensity of bleomycin damage sites in the LCR HS-2 was f ound to differ substantially between intact cells and purified DNA at putative transcription factor binding sites. Bleomycin activity was gr eatly reduced in cells at a tandem NF-E2/AP1 DNA sequence element. Thi s footprint was strongest in K562 cells where the nuclear factor-eryth roid 2 (NF-E2) is thought to bind. Protection and enhancement were als o observed at other sequence elements in the HS-2 that associate with erythroid-specific and ubiquitous transcription factors. These results suggest that the activity of bleomycin is significantly reduced at th e site of protein-DNA interactions in intact cells. This property of b leomycin is extremely useful in genomic ''footprinting'', where it has significant advantages over other commonly used agents.