Asymmetric recognition of psoralen interstrand crosslinks by the nucleotide excision repair and the error-prone repair pathways

Psoralen is an asymmetric photoreactive intercalator with a furane and a py rone side. When intercalated at 5'-TpA-3' sites and upon UVA irradiation, t he psoralen can react with the thy-nine residues on both strands, introduci ng an interstrand crosslink. Using psoralen-coupled triple-helix-forming ol igonucleotides, psoralen interstrand crosslinks can be site-specifically in troduced in the coding sequence of URA3, a yeast auxotrophic marker carried on plasmid vectors. Ln addition, crosslinks introduced via a triple-helix- forming oligonuleotide are oriented with the furane side of the psoralen as sociated with a specific strand of the target sequence. Here, the transform ation efficiency, the mutation frequency and the mutational spectra of site -specifically placed and oriented cross-links were examined in yeast cells. We found that the nature of the targeted mutations depended on the crossli nk orientation: bypass of the pyrone-adducted thymine yielded T --> A or T --> C substitutions and A insertions, while bypass of the furane-adducted t hymine yielded T --> G substitutions and G insertions. Thus, the structure of the damage strongly influences the choice of the nucleotide incorporated during translesion synthesis. Ln addition, the observed pattern of mutagen esis suggests a coupling to transcription, similar to the one observed in m ammalian cells. Finally, the substitutions affected only the coding strand when the pyrone link of the psoralen crosslink was on this strand, whereas they affected both strands when the pyrone link was on the transcribed stra nd, suggesting that the incision preference of psoralen crosslinks, which h as been observed with purified uvrABC proteins in bacteria, is conserved in live eucaryotic cells. (C) 1999 Academic Press.