In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction.
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.
Pfeifer
G P
GP
Beckman Research Institute of the City of Hope, Department of Biology, Duarte, CA 91010.
Drouin
R
R
Riggs
A D
AD
Holmquist
G P
GP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000528:radiation effects*; D006801:Humans; D006822:Hybrid Cells / Q000528:radiation effects; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D011740:Pyrimidine Dimers / Q000032:analysis*; D014466:Ultraviolet Rays; D014960:X Chromosome / Q000528:radiation effects
D015345:Oligonucleotide Probes; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; D004247:DNA; D010735:Phosphoglycerate Kinase