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Published February 12, 2008 | Supplemental Material
Journal Article Open

DNA Oxidation by Charge Transport in Mitochondria


Sites of oxidative DNA damage in functioning mitochondria have been identified using a rhodium photooxidant as a probe. Here we show that a primer extension reaction can be used to monitor oxidative DNA damage directly in functioning mitochondria after photoreaction with a rhodium intercalator that penetrates the intact mitochondrial membrane. The complex [Rh(phi)_2bpy]Cl_3 (phi = 9,10-phenanthrenequinonediimine) binds to DNA within the mitochondria and, upon irradiation, initiates DNA oxidation reactions. Significantly, piperidine treatment of the mitochondria leads to protein-dependent primer extension stops spaced every ∼20 base pairs. Hence, within the mitochondria, the DNA is well covered and packaged by proteins. Photolysis of the mitochondria containing [Rh(phi)_2bpy]^(3+) leads to oxidative DNA damage at positions 260 and 298; both are mutational hot spots associated with cancers. The latter position is the 5'-nucleotide of conserved sequence block II and is critical to replication of the mitochondrial DNA. The oxidative damage is found to be DNA-mediated, utilizing a charge transport mechanism, as the Rh binding sites are spatially separated from the oxidation-prone regions. This long-range DNA-mediated oxidation occurs despite protein association. Indeed, the oxidation of the mitochondrial DNA leads not only to specific oxidative lesions, but also to a corresponding change in the protein-induced stops in the primer extension. Mitochondrial DNA damage promotes specific changes in protein−DNA contacts and is thus sensed by the mitochondrial protein machinery.

Additional Information

© 2008 American Chemical Society. Received August 30, 2007; Revised Manuscript Received November 20, 2007. Publication Date (Web): January 12, 2008. We are grateful to the NIH (Grant GM49216) for their financial support of this research including a minority postdoctoral fellowship to E.J.M.

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