Influence of Chromatin Structure on Bleomycin-DNA Interactions at Base Pair Resolution in the Human -Globin Gene Clus
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- 作者：Murray J. Cairns and Vincent Murray
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：July 2, 1996
- 年：1996
- 卷：35
- 期：26
- 页码：8753 - 8760
- 全文大小：393K
- 年卷期：v.35,no.26(July 2, 1996)
- ISSN：1520-4995
文摘
The DNA sequence specificity of bleomycin was examinedin human cells and in purifiedgenomic DNA. In each case, DNA damage sites were determined atnucleotide resolution in the humansingle-copy 
-globin promoter and the locus control region (LCR)hypersensitive site 2 (HS-2). Exponentialamplification of gene-specific genomic fragments was achieved byligation-mediated PCR, and labeledreaction products were analyzed directly by sequencing gelelectrophoresis. Bleomycin was found tocleave DNA preferentially at GC, GT, and GA dinucleotides. Thisstudy represents the first occasionthat the sequence specificity of bleomycin has been determined inintact human cells at the single-copygene level. The intensity of bleomycin damage sites in the LCRHS-2 was found to differ substantiallybetween intact cells and purified DNA at putative transcription factorbinding sites. Bleomycin activitywas greatly reduced in cells at a tandem NF-E2/AP1 DNA sequenceelement. This footprint was strongestin K562 cells where the nuclear factor-erythroid 2 (NF-E2) is thoughtto bind. Protection and enhancementwere also observed at other sequence elements in the HS-2 thatassociate with erythroid-specific andubiquitous transcription factors. These results suggest that theactivity of bleomycin is significantly reducedat the site of protein-DNA interactions in intact cells. Thisproperty of bleomycin is extremely usefulin genomic "footprinting", where it has significant advantages overother commonly used agents.
-globin promoter and the locus control region (LCR)hypersensitive site 2 (HS-2). Exponentialamplification of gene-specific genomic fragments was achieved byligation-mediated PCR, and labeledreaction products were analyzed directly by sequencing gelelectrophoresis. Bleomycin was found tocleave DNA preferentially at GC, GT, and GA dinucleotides. Thisstudy represents the first occasionthat the sequence specificity of bleomycin has been determined inintact human cells at the single-copygene level. The intensity of bleomycin damage sites in the LCRHS-2 was found to differ substantiallybetween intact cells and purified DNA at putative transcription factorbinding sites. Bleomycin activitywas greatly reduced in cells at a tandem NF-E2/AP1 DNA sequenceelement. This footprint was strongestin K562 cells where the nuclear factor-erythroid 2 (NF-E2) is thoughtto bind. Protection and enhancementwere also observed at other sequence elements in the HS-2 thatassociate with erythroid-specific andubiquitous transcription factors. These results suggest that theactivity of bleomycin is significantly reducedat the site of protein-DNA interactions in intact cells. Thisproperty of bleomycin is extremely usefulin genomic "footprinting", where it has significant advantages overother commonly used agents.