Pfeifer G P, Drouin R, Riggs A D, Holmquist G P
Beckman Research Institute of the City of Hope, Department of Biology, Duarte, CA 91010.
Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1374-8. doi: 10.1073/pnas.88.4.1374.
DNA adducts in unique sequences along the mammalian genome are mapped in vivo at single-nucleotide resolution. Pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts] represent one of the two major adduct classes found after UV irradiation of DNA and were shown to play an important role in UV-induced mutagenesis. After UV light treatment of cells, DNA is prepared and chemically cleaved at (6-4) photoproducts with piperidine. Gene-specific fragments are then amplified from total genomic DNA by use of a ligation-mediated polymerase chain reaction. Analysis of the human chromosome X-linked phosphoglycerate kinase (PGK1) gene's promoter has shown that the frequency of (6-4) photoproducts expressed as piperidine-labile sites is (i) high at TpC and CpC dinucleotides, (ii) dependent on the nearest-neighbor bases, (iii) inhibited by the binding of a transcription factor, and (iv) different for DNA derived from the active and inactive X chromosome. This latter difference is mainly a consequence of the presence of 5-methylcytosine (m5C) in CpG dinucleotides on the inactive X chromosome. 5-Methylcytosine in the sequences Tm5CG and Cm5CG inhibits the formation of (6-4) photoproducts. Thus, in addition to in vivo mapping of a DNA adduct at nucleotide resolution, we also report another method for methylation analysis and photofootprinting.
沿着哺乳动物基因组独特序列的DNA加合物在体内以单核苷酸分辨率进行定位。嘧啶（6-4）嘧啶酮光产物[（6-4）光产物]是DNA紫外线照射后发现的两种主要加合物类型之一，并已证明在紫外线诱导的诱变中起重要作用。细胞经紫外线处理后，制备DNA并使用哌啶在（6-4）光产物处进行化学切割。然后通过连接介导的聚合酶链反应从总基因组DNA中扩增基因特异性片段。对人类X染色体连锁磷酸甘油酸激酶（PGK1）基因启动子的分析表明，以哌啶不稳定位点表示的（6-4）光产物频率为：（i）在TpC和CpC二核苷酸处较高；（ii）取决于最近邻碱基；（iii）受转录因子结合抑制；（iv）来自活性和非活性X染色体的DNA不同。后一种差异主要是由于非活性X染色体上CpG二核苷酸中存在5-甲基胞嘧啶（m5C）。序列Tm5CG和Cm5CG中的5-甲基胞嘧啶抑制（6-4）光产物的形成。因此，除了以核苷酸分辨率对DNA加合物进行体内定位外，我们还报告了另一种甲基化分析和光足迹法。
