DNA-mediated signaling by proteins with 4Fe-4S clusters is necessary for genomic integrity

Abstract

Iron-sulfur clusters have increasingly been found to be associated with enzymes involved in DNA processing. Here we describe a role for these redox clusters in DNA-mediated charge-transport signaling in E. coli between DNA repair proteins from distinct pathways. DNA-modified electrochemistry shows that the 4Fe-4S cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III (EndoIII), a base excision repair enzyme, cooperate at long-range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Silencing the gene encoding EndoIII in a strain of E. coli where repair by DinG is essential results in a significant growth defect that is rescued by complementation with EndoIII but not with an EndoIII mutant that is enzymatically active but unable to carry out DNA charge transport. This work thus elucidates a fundamental mechanism to coordinate the activities of DNA repair enzymes across the genome.

Figure 1

Electrochemistry of DinG on DNA-modified electrodes. (A) Cyclic voltammogram of 10 μM DinG (red), DinG after the addition of 5 mM ATP (blue), and buffer only (black) after incubation for 3 h. Inset: Cartoon representation of a protein bound to DNA on a DNA-modified electrode. (B) Percent change in current after the addition of 1 mM ATP (blue) or 1 mM ATPγs (black). Percent change in current is the percent increase in the measured current compared to the predicted current, based on the linear growth of the signal with respect to time for the incubation of DinG before the addition of ATP.

Figure 2

AFM redistribution assay. (A) A flattened image (Bruker nanoscope analysis software) for tapping-mode AFM topography of DinG-bound DNA adsorbed on mica. (B) Schematic representation of the redistribution assay. At equilibrium, repair proteins (blue) are preferentially localized on strands of DNA (black) with a C:A mismatch (red X). (C) Three-dimensional rendering of the blue-bordered region of the AFM image in A that shows a strand of DNA bound by two DinG proteins. (D) Measured binding density ratios, the density of proteins on long strands divided by the density of proteins on short strands, for proteins bound to mixtures of long and short strands of DNA with and without a mismatch (C:A) in the middle of the long strand. Three separate mixtures of protein and DNA were deposited onto individual surfaces, and at least 50 images were analyzed for each DinG (blue), a mixture of DinG and EndoIII (red), and a mixture of DinG and a CT-deficient mutant, Y82A EndoIII (green). ± SEM using a single image as a data point.

Figure 3

Rescue of growth defect conferred by knocking out nth in InvA. Cultures of LB were inoculated with single colonies of each strain and growth was monitored by optical density at 600 nm over time. Strains of InvA Δnth grew comparably to InvA Δnth transformed with p(empty) showing that the effect is not due to the presence of the plasmid. Data were recorded for at least three independent trials. (A) Growth of InvA WT (blue) or InvA Δnth transformed with p(empty) (red) ± SEM. (B) Growth of InvA Δnth transformed with p(WT EndoIII) (blue) or p(empty) (red) ± SEM. (C) Growth of InvA Δnth transformed with p(RNaseH) (blue) or p(empty) (red) ± SEM. (D) Growth of InvA Δnth transformed with p(EndoIII D138A) (blue) or p(empty) (red) ± SEM. (E) Growth of InvA Δnth transformed with p(EndoIII Y82A) (black) or p(empty) (red) ± SEM.

Figure 4

Scheme depicting how repair proteins may use DNA-mediated signaling to search for damage. The model describes how DNA CT can drive the redistribution of the repair proteins into the vicinity of damage. (1) A protein with a reduced (orange-yellow) iron–sulfur cluster binds to DNA. (2) This protein’s iron–sulfur cluster is oxidized (purple-brown) by another DNA-bound redox-active protein. This oxidation can occur over long distances and through other DNA-bound proteins (gray) so long as the π-orbital stacking of bases between the reductant and oxidant is unperturbed. (3) Reduction promotes the repair protein’s dissociation from DNA. (4) The repair protein binds to an alternate DNA site where it is oxidized either by a guanine radical or another protein. (5) DNA lesions between proteins inhibit electron transport, so protein dissociation is not promoted. (6) Proteins that are now in close proximity to the lesion are able to move processively toward the damage for repair.