Mutants of the Base Excision Repair Glycosylase, Endonuclease III: DNA Charge Transport as a First Step in Lesion Detection

Abstract

Endonuclease III (EndoIII) is a base excision repair glycosylase that targets damaged pyrimidines and contains a [4Fe-4S] cluster. We have proposed a model where BER proteins that contain redox-active [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in the detection of DNA lesions. Here, several mutants of EndoIII were prepared to probe their efficiency of DNA/protein charge transport. Cyclic voltammetry experiments on DNA-modified electrodes show that aromatic residues F30, Y55, Y75 and Y82 help mediate charge transport between DNA and the [4Fe-4S] cluster. Based on circular dichroism studies to measure protein stability, mutations at residues W178 and Y185 are found to destabilize the protein; these residues may function to protect the [4Fe-4S] cluster. Atomic force microscopy studies furthermore reveal a correlation in the ability of mutants to carry out protein/DNA CT and their ability to relocalize onto DNA strands containing a single base mismatch; EndoIII mutants that are defective in carrying out DNA/protein CT do not redistribute onto mismatch-containing strands, consistent with our model. These results demonstrate a link between the ability of the repair protein to carry out DNA CT and its ability to relocalize near lesions, thus pointing to DNA CT as a key first step in the detection of base damage in the genome.

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

“…We have previously demonstrated with BER proteins that the ability of a protein to perform DNA CT directly correlates to its redistribution in the vicinity of base lesions or mismatches that inhibit CT (23). We tested XPD redistribution promoted through DNA CT by preparing DNA strands containing a single C∶A mismatch, a modification that we know to inhibit CT (40), alongside DNA strands containing no mismatches.…”

“…To prepare these DNA samples, pUC19 plasmid was amplified with primers incorporating a 2′ O-methyl residue to yield two short duplexes containing 14-nucleotide single-strand overhangs (23,41). Upon ligation of these two duplexes, 3.8 kbp strands were formed that contained only one C∶A mismatch in the middle of the strand.…”

“…We have recently examined whether DNA CT may play some role in how these BER proteins find their site (22,23). Using DNA electrochemistry and atomic force microscopy (AFM) experiments, we found that the ability of EndoIII mutants to localize in the vicinity of a base mismatch correlates with their ability to carry out DNA CT.…”

“…Thus, in the model, DNA-mediated reduction of one protein by the other leads to the dissociation of the reduced protein from DNA, effectively giving overall dissociation of repair proteins away from a region of the genome without lesions. Our model relies on the sensitivity of CT to proper π stacking of the bases between the donor protein and the distant acceptor protein (22,23); if instead there is an intervening lesion, DNA-mediated CT does not occur, and the repair protein remains bound and can processively move to the lesion and carry out enzymatic repair. In this process, the repair proteins eventually relocalize in the vicinity of lesions or any modification that inhibits DNA CT. As a result the overall search regime for the repair proteins is made smaller than the full genome: they preferentially localize where needed for repair.…”

“…Alternatively, if enzymes rely on DNA/ protein CTas a first step in finding damage, then any protein that can carry out DNA CT can participate in the search, integrating repair pathways that were previously thought to be separate. To examine this coordinated search, AFM is used to visualize mixtures containing two types of proteins: (i) those that are able to carry out DNA CT and (ii) those that are defective at DNA CT. AFM, a technique that allows for visualization of single molecule protein/DNA interactions, can be used to observe proteins bound to long strands (3.8 kbp) of DNA containing a site-specific mismatch (C∶A) (23,(36)(37)(38)(39). Binding density ratios calculated from counting strands and proteins in AFM images can then give us clues to the first step in lesion detection.…”