DNA charge transport as a first step in coordinating the detection of lesions by repair proteins

Abstract

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. We have proposed a model where repair proteins containing redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5'-3' helicase involved in nucleotide excision repair, contains a [4Fe-4S] cluster and exhibits a DNA-bound redox potential that is physiologically relevant. In AFM studies, we observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. We further provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster. When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand. However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs. These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome.

Fig. 1.

Model for damage detection and redistribution by repair proteins. (Top) Given two repair proteins, containing [4Fe-4S] clusters, that are able to carry out DNA CT, such as XPD or EndoIII (blue), CT can proceed through well matched unmodified DNA. Driven by protein oxidation by guanine radicals (yellow) formed under oxidative stress, DNA CT occurs between the DNA-bound proteins, promoting the dissociation of reduced protein (light blue). DNA CT does not occur, however, in the presence of an intervening mismatch or lesion (red X); with the intervening mismatch or lesion, repair proteins are not reduced from a distance, and therefore do not dissociate but instead remain bound in the vicinity of the lesion. (Bottom) If repair proteins are mutants such as XPD L325V or EndoIII Y82A (red) and are unable to perform DNA CT (dashed line), they cannot send or receive signals to locate damage. In the presence of a lesion, these proteins then do not preferentially redistribute in the vicinity of the mismatch.

Fig. 2.

AFM to visualize DNA-bound proteins. Tapping mode AFM images of DNA and XPD protein on mica imaged in air. The matched strands and protein (dots) are visible on the surface (top). Because the single strand overhangs on the short duplexes have been blocked by annealing short oligomers, XPD is bound in random positions on the DNA. Zoomed-in images of long (3.8 kbp) and short (1.6 and 2.2 kbp) DNA strands with bound proteins (black arrows) are shown below. Images were acquired with a scan size of 2 × 2 μm² or 3 × 3 μm², at a rate of 3.05 Hz with a data scale of 10 nm/0.5 V.

Fig. 3.

DNA CT with the L325V mutant. (Left) Cyclic Voltammogram (CV) of WT SaXPD [8 μM] (green) and L325V SaXPD [8 μM] (red) on DNA-modified electrodes after 90 min. (Right) Quantitation of XPD and mutant L325V protein density ratios (< 10% uncertainty) where C∶A indicates a mismatch is contained in long strands. The unmarked bars show the control measurements for fully matched long and short strands. XPD redistributes onto mismatched strands. L325V, CT deficient, does not show redistribution.

Fig. 4.

Representative tapping mode AFM images. Fully matched long and short DNA strands (left) or mismatched long DNA and matched short DNA strands (right) are incubated overnight with XPD/EndoIII 1∶1 protein mixture. Inset shows zoomed-in view of a long DNA strand with both large (approximately 6 nm) and small (approximately 3.5 nm) proteins bound.

Fig. 5.

Summary of binding density ratios for XPD (red text)/EndoIII (blue text) and mutant mixtures. Quantitation of protein density ratios (< 10% uncertainty) where C∶A indicates a mismatch is contained in long strands. WT^XPD/WT^EndoIII mixtures 1∶1 (purple), both proteins CT proficient, redistribute onto mismatched strands. WT^XPD/Y82A^EndoIII and WT^EndoIII/L325V^XPD 1∶1 mixtures (green and orange respectively), Y82A and L325V both mutants deficient in CT, do not show redistribution. WT^XPD/L325V^XPD 1∶1 mixtures (blue) do not show redistribution, as XPD cannot perform DNA-mediated signaling if the L325V mutant is present.