Redox signaling between DNA repair proteins for efficient lesion detection

Abstract

Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and damaged base pairs, we have proposed that DNA repair glycosylases (EndoIII and MutY) containing a redox-active [4Fe4S] cluster could use DNA CT in signaling one another to search cooperatively for damage in the genome. Here, we examine this model, where we estimate that electron transfers over a few hundred base pairs are sufficient for rapid interrogation of the full genome. Using atomic force microscopy, we found a redistribution of repair proteins onto DNA strands containing a single base mismatch, consistent with our model for CT scanning. We also demonstrated in Escherichia coli a cooperativity between EndoIII and MutY that is predicted by the CT scanning model. This relationship does not require the enzymatic activity of the glycosylase. Y82A EndoIII, a mutation that renders the protein deficient in DNA-mediated CT, however, inhibits cooperativity between MutY and EndoIII. These results illustrate how repair proteins might efficiently locate DNA lesions and point to a biological role for DNA-mediated CT within the cell.

Fig. 1.

A model for DNA-mediated CT in DNA repair. In this model, DNA repair proteins containing [4Fe4S]²⁺ clusters—for example, EndoIII (green) and MutY (orange)—bind DNA, activating them toward oxidation to the [4Fe4S]³⁺ state. The sequence of events is as follows: Guanine radical formation can oxidize a repair protein in a DNA-mediated reaction, stabilizing the oxidized protein bound to DNA (A). A second protein binds near the first protein (B and E). CT to a distally bound protein can occur if the intervening DNA is undamaged (C and F). The newly reduced protein has a diminished affinity for DNA and diffuses away (D). If a lesion site is present between the proteins (G), the DNA-mediated CT step is inhibited, and the oxidized protein remains bound. The sum of the DNA-mediated CT steps between proteins constitutes a full search of the genome.

Fig. 2.

Scanning time as a function of maximum distance of DNA-mediated interprotein CT (N) and the fraction of repair proteins that are in the 3+ state (% ox) by using the CT scanning model. At 10% oxidized protein with a maximum CT distance of 500 bp, the time required to interrogate the genome is ≈5 min.

Fig. 3.

Measurements of repair protein distributions on DNA by AFM. A zoomed-in view (Left) of representative AFM images of DNA strands incubated overnight with WT EndoIII. A higher density of proteins is apparent on the longer DNA strands containing the single CA mismatch. (Right) Quantitation of protein density ratios (<10% uncertainty). A CA mismatch is contained on the long strand except for the sample indicated by matched DNA, where both the long and the short strands are fully matched.

Fig. 4.

Y82A EndoIII, a mutant in DNA-mediated CT capability. (A) Bar graph showing lac⁺ revertants for CC104/p, CC104 nth⁻/p, CC104/Y82A, and CC104 nth⁻/Y82A strains, where p denotes inclusion of an empty vector. Lac⁺ revertants are reported as the average number of lac⁺ colonies that arise per 10⁹ cells plated on minimal lactose medium containing ampicillin. Data for the CC104 strains are shown based on five sets of independent experiments, each containing 10 replicates per strain. (B) Autoradiogram after denaturing PAGE of ³²P-5′-TGTCAATAGCAAGXGGAGAAGTCAATCGTGAGTCT-3′ plus complementary strand, where X = 5-OH-dU base-paired with G. Protein samples (100 or 10 nM) were incubated with duplexes for 15 min at 37 °C and quenched with 1 M NaOH. Cleavage of the ³²P-labeled strand at the lesion site (X) by EndoIII results in formation of a 14-mer. No significant difference in glycosylase activity (10% uncertainty) is observed between Y82A and WT EndoIII. (C) Cyclic voltammetry of Y82A EndoIII at an Au electrode modified with SH(CH₂)₂CONH(CH₂)₆NHOCO-5′-AGTACAGTCATCGCG-3′ plus complementary strand showing the reduction and reoxidation of the DNA-bound protein. DNA-modified surfaces were prepared and backfilled with mercaptohexanol, and WT or Y82A EndoIII was tested. Surfaces were then rinsed, and the other protein was analyzed on the same surface. Over several trials, the electrochemical signal associated with Y82A was 50% ± 13% smaller per [4Fe4S] cluster compared with WT EndoIII, reflecting poor electronic coupling of the mutant to the DNA-modified electrode. (D) Comparative densities for WT (Left) and Y82A (Right) EndoIII bound to matched versus mismatched (CA) strands measured by AFM. Although WT EndoIII preferentially redistributes onto the mismatched strand, Y82A shows no preference.