标题：In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction.
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1991
卷号：88
期号：4
页码：1374-1378
DOI：10.1073/pnas.88.4.1374
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
