An Anionic Diplatinum DNA Photocleavage Agent: Chemical Mechanism and Footprinting of .lambda. Repressor

The d sigma * arrow right p sigma excited state of Pt sub(2)(pop) sub(4) super(4-) (1, pop = P sub(2)O sub(5)H sub(2) super(2-)) elicits frank scission of double-stranded DNA as assayed by high-resolution gel electrophoresis. The photoreaction of 1 and a 5'- super(32)P-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) super(2-)/H sub(2)O sub(2) reaction. The analogous reaction of the 3'- super(32)P-labeled duplex also produces phosphate termini and a modified band that can be assigned as a 5'-aldehyde terminus by NaBH sub(4) 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 sub(2), which appears to trap the radical produced by homolytic C-H activation. The even cleavage ladder argues strongly against a super(1)O sub(2) mechanism, and the cleavage is not enhanced in D sub(2)O. 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 Mg super(2+). This unusual reaction has been used to obtain a footprint of lambda repressor bound to the O sub(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.