Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

Abstract

The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. We took advantage of the blockage by bulky adducts of the 3''-5''-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T < TT .mchlt. TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT .mchlt. TA .apprx. TAT .mchlt. < ATAT < ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5''-TpA sites. Our results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA.