Cross-linking of DNA-binding Proteins to DNA with Psoralen and Psoralen Furan-side Monoadducts

Abstract

We have developed a novel photocross-linking tech- nique using free 8-methoxypsoralen andDNAfuran-side monoadducts plus long wave ultraviolet light (UVA). Both sequence-specific (Max) and nonspecific (RecA and T7 RNA polymerase) DNA-binding proteins were cross- linked. The macroscopic equilibrium binding constant (109 M1) and DNase I footprinting indicated that bind- ing of Max to its cognate sequence (E-box) was unim- paired by 8-methoxypsoralen and that cross-linking oc- curred in normal complexes. RecA protein and T7 RNA polymerase were cross-linked to a 12-mer DNA furan- side monoadduct with UVA. Cross-link yields were di- rectly proportional to the UVA dose. Cross-links were stable to 8 M urea, 1–10% SDS, commonly used alcohols, and mild acids (5% trichloroacetic acid). The DNA in cross-links was reversed with 254 nm UV (photorever- sal) or with hot base (base-catalyzed reversal), consist- ent with (2  2) cycloaddition via the 4,5-furan of the psoralen. Comparative action spectra for DNA-DNA cross-linking and DNA-protein cross-linking revealed that the latter occurred maximally at 300 nm, while the former occurred maximally at 320 nm. This 20-nm blue shift suggested a higher potential energy surface for an excited psoralen participating in protein-DNA cross- linking as compared with DNA-DNA cross-linking. As with DNA-DNA cross-linking, DNA-protein cross-linking is a two-photon process. Absorption of the first photon formed a 4´,5-adduct with DNA, which then absorbed a second photon, leading to cross-linking to protein. Based on the action spectra and the known excited states of psoralen, it is suggested that the triplet n, * transition localized in the C-2O of psoralen may be involved in protein-psoralen photoreactions.