8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.
We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand.
Sage
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CNRS URA 1292, Institut Curie-Section de Biologie, Paris, France.
Drobetsky
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EA
Moustacchi
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D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects*; D008730:Methoxsalen / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology*; D010777:Photochemistry; D013941:Thymine / Q000378:metabolism; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D009153:Mutagens; D004247:DNA; D000228:Adenine Phosphoribosyltransferase; D013941:Thymine; D008730:Methoxsalen