DNA-Bound Redox Activity of DNA Repair Glycosylases Containing [4Fe-4S] Clusters

Abstract

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…If CT to the cluster is mediated by the DNA π-stack, then, the introduction of an abasic site into the duplex would be expected to attenuate the signal from the [4Fe-4S] cluster. Indeed, both MutY and Endo III exhibit a diminished signal at an electrode modified with DNA containing the intervening abasic site [55][56]. This result demonstrates that CT through the π-stack of DNA is required to observe [4Fe-4S] cluster oxidation electrochemically.…”

“…MutY and Endo III both exhibit a quasi-reversible signal when investigated with cyclic voltammetry at a DNA-modified Au electrode [55][56]. This signal is not observed at a mercaptohexanol-modified Au electrode.…”

“…DNA-modified electrodes have also been shown to be useful in studying DNA-binding proteins that contain their own redox cofactor, such as MutY and Endo III [55][56][57]. In these experiments, modified DNA is self-assembled into a loosely packed monolayer and backfilled with an alkane or alkanethiol moiety to prevent any nonspecific interactions between the protein and the electrode surface (Figure 1).…”

“…To test the importance of DNA-mediated CT in the redox activity of the [4Fe-4S] cluster, we evaluated MutY and Endo III on an electrode containing a duplex with an abasic site incorporated only three bases away from the electrode surface [55][56]. Previous studies indicate that an abasic site drastically attenuates CT to a redox active intercalator bound above the lesion site [48].…”

“…A cationic oxidant, Co(phen) 3 3+ (phen = 1,10-phenanthroline), that binds DNA, was employed to determine whether binding of the protein to DNA might promote oxidation of the [4Fe-4S] 2+ cluster in solution [56]. When Endo III is incubated with three molar equivalents Co(phen) 3 3+ , a small signal is observed that is typical of the [3Fe-4S] 1+ cluster.…”