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

Abstract

We have developed a novel photocross-linking technique using free 8-methoxypsoralen and DNA furan-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 ( approximately 10(9) M-1) and DNase I footprinting indicated that binding of Max to its cognate sequence (E-box) was unimpaired by 8-methoxypsoralen and that cross-linking occurred 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 directly 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 (photoreversal) or with hot base (base-catalyzed reversal), consistent 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,pi* transition localized in the C-2=O of psoralen may be involved in protein-psoralen photoreactions.