AN ANIONIC DIPLATINUM DNA PHOTOCLEAVAGE AGENT - CHEMICAL MECHANISM AND FOOTPRINTING OF LAMBDA-REPRESSOR

The d sigma* --> p sigma excited state of Pt-2(pop)(4)(4-) (1, pop = P2O5H22-) elicits frank scission of double-stranded DNA as assayed by high-resolution gel electrophoresis. The photoreaction of 1 and a 5'-P-32-labeled 25-mer duplex produces a surprisingly even ladder of phosphate terminated bands with some modified bands that can be assigned as phosphoglycolate termini by comigration with the products of an Fe(EDTA)(2)-/H2O2 reaction. The analogous reaction of the 3'-P-32-labeled duplex also produces phosphate termini and a modified band that can be assigned as a 5'-aldehyde terminus by NaBH4 reduction to the 5'-alcohol and comigration with authentic alcohol termini generated using alkaline phosphatase. These products are consistent with abstraction of the 4' and 5' hydrogens from the deoxyribose function; products indicative of 1' or 3' chemistry were not detected. The reaction is more efficient in the presence of O-2, which appears to trap the radical produced by homolytic C-H activation. The even cleavage ladder argues strongly against a O-1(2) mechanism, and the cleavage is not enhanced in D2O. Further, ethanol does not inhibit the reaction of 1 at concentrations up to 1 M, where the reaction of hydroxyl radical is completely quenched. These experiments point to a mechanism where the tetraanionic complex collides directly with the DNA to effect C-H activation, which is supported by a strong enhancement in cleavage by Mg2+. This unusual reaction has been used to obtain a footprint of lambda repressor bound to the O(R)1 sequence. The resolution of the footprint is similar to that of hydroxyl radical, which permits binding of the repressor to a single side of the DNA helix to be distinguished.