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 investigated in the single-copy human beta-globin gene cluster in an erythroid cell line, a nonerythroid cell line, and purified genomic DNA. The target DNA sequences for this study were the beta-globin gene locus control region (LCR) hypersensitive site 2 (HS-2) and the beta-globin gene promoter. The DNA fragments produced by HDM damage in these target sequences were selectively amplified by the ligation-mediated polymerase chain reaction (LMPCR) and analyzed at nucleotide resolution by DNA-sequencing gel electrophoresis. The DNA sequences damaged by HDM in the cellular environment were found to be similar to that observed in the purified genomic DNA. However, substantial differences did occur between the intensity of cellular and purified genomic DNA reaction products at discrete regions corresponding to transcription factor-binding motifs. 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. This motif has been shown to bind to the erythroid-specific nuclear factor-erythroid 2 (NF-E2) and the widely distributed activator protein-1 (AP-1). The HDM damage protection patterns or "genomic footprints" observed 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 damaging activity is modulated by protein-DNA interactions, it may have application in genomic footprinting experiments.
Cairns
M J
MJ
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.
Murray
V
V
D000477:Alkylating Agents / Q000494:pharmacology*; D000880:Anthraquinones / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects* / Q000235:genetics; D004249:DNA Damage; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D019901:Locus Control Region / Q000235:genetics; D005810:Multigene Family; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000477:Alkylating Agents; D000880:Anthraquinones; D014157:Transcription Factors; C001328:hedamycin; D005914:Globins; D004247:DNA