Cairns M J, Murray V
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.
Biochemistry. 1996 Jul 2;35(26):8753-60. doi: 10.1021/bi9600207.
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.
在人类细胞和纯化的基因组DNA中检测了博来霉素的DNA序列特异性。在每种情况下，均在人类单拷贝β-珠蛋白启动子和基因座控制区（LCR）超敏位点2（HS-2）以核苷酸分辨率确定DNA损伤位点。通过连接介导的PCR实现基因特异性基因组片段的指数扩增，并通过测序凝胶电泳直接分析标记的反应产物。发现博来霉素优先在GC、GT和GA二核苷酸处切割DNA。这项研究是首次在完整的人类细胞中单拷贝基因水平上确定博来霉素的序列特异性。发现在完整细胞和纯化DNA中，假定转录因子结合位点处LCR HS-2中的博来霉素损伤位点强度存在显著差异。在串联的NF-E2/AP1 DNA序列元件处，细胞中的博来霉素活性大大降低。该足迹在K562细胞中最强，据认为核因子红细胞2（NF-E2）在该细胞中结合。在与红细胞特异性和普遍存在的转录因子相关的HS-2中的其他序列元件处也观察到了保护和增强作用。这些结果表明，在完整细胞中，蛋白质-DNA相互作用位点处博来霉素的活性显著降低。博来霉素的这一特性在基因组“足迹分析”中极为有用，与其他常用试剂相比具有显著优势。
