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

The dσ* → pσ excited state of Pt2(pop)4^4– (1, pop = P2O5H2^2–) elicits frank scission of double-stranded DNA as assayed by high-resolution gel electrophoresis. The photoreaction of 1 and a 5′-³²P-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′-³²P-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 O2, which appears to trap the radical produced by homolytic C–H activation. The even cleavage ladder argues strongly against a ¹O2 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 λ repressor bound to the OR1 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. © 1995, American Chemical Society. All rights reserved.

Duke Scholars

Author Terrence Gilbert Oas Biochemistry