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Biochemistry. 1996 Jul 2;35(26):8753-60.

Influence of chromatin structure on bleomycin-DNA interactions at base pair resolution in the human beta-globin gene cluster.

Author information

1
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.

Abstract

The DNA sequence specificity of bleomycin was examined in human cells and in purified genomic DNA. In each case, DNA damage sites were determined at nucleotide resolution in the human single-copy beta-globin promoter and the locus control region (LCR) hypersensitive site 2 (HS-2). Exponential amplification of gene-specific genomic fragments was achieved by ligation-mediated PCR, and labeled reaction products were analyzed directly by sequencing gel electrophoresis. Bleomycin was found to cleave DNA preferentially at GC, GT, and GA dinucleotides. This study represents the first occasion that the sequence specificity of bleomycin has been determined in intact human cells at the single-copy gene level. The intensity of bleomycin damage sites in the LCR HS-2 was found to differ substantially between intact cells and purified DNA at putative transcription factor binding sites. Bleomycin activity was greatly reduced in cells at a tandem NF-E2/AP1 DNA sequence element. This footprint was strongest in K562 cells where the nuclear factor-erythroid 2 (NF-E2) is thought to bind. Protection and enhancement were also 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 the site of protein-DNA interactions in intact cells. This property of bleomycin is extremely useful in genomic "footprinting", where it has significant advantages over other commonly used agents.

PMID:
8679639
DOI:
10.1021/bi9600207
[Indexed for MEDLINE]

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