Cross-linking of DNA-binding proteins to DNA with psoralen and psoralen furan-side monoadducts. Comparison of action spectra with DNA-DNA cross-linking.
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.
Sastry
S S
SS
Laboratory of Molecular Genetics, and the Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA. sastrys@rockvax.rockefeller.edu
Ross
B M
BM
P'arraga
A
A
D050976:Basic-Leucine Zipper Transcription Factors; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005363:Ficusin / Q000494:pharmacology*; D008730:Methoxsalen / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D014157:Transcription Factors; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000494:pharmacology
D050976:Basic-Leucine Zipper Transcription Factors; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C067850:Myc associated factor X; D014157:Transcription Factors; C016507:hydroxymethyltrioxsalen; D004247:DNA; D011938:Rec A Recombinases; D005363:Ficusin; D008730:Methoxsalen; D014307:Trioxsalen