DNA Charge Transport within the Cell

The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long-range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long-range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor the integrity of the DNA, given the sensitivity of DNA CT to perturbations in base stacking as arise with mismatches and lesions. Enzymes that utilize this chemistry include an interesting and ever-growing class of DNA-processing enzymes involved in DNA repair, replication, and transcription that have been found to contain 4Fe-4S clusters. DNA repair enzymes containing 4Fe-4S clusters, that include endonuclease III (EndoIII), MutY, and DinG from bacteria, as well as XPD from archaea, have been shown to be redox-active when bound to DNA, share a DNA-bound redox potential, and can be reduced and oxidized at long-range via DNA CT. Interactions between DNA and these proteins in solution, in addition to genetics experiments within Escherichia coli, suggest that DNA-mediated CT can be used as a means of cooperative signaling among DNA repair proteins that contain 4Fe-4S clusters as a first step in finding DNA damage, even within cells. On the basis of these data, we can consider also how DNA-mediated CT may be used as a means of signaling to coordinate DNA processing across the genome

DNA Charge Transport: from Chemical Principles to the Cell

The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology

UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC

UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC

Multiplexed Electrochemistry of DNA-Bound Metalloproteins

Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of endonuclease III (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster

DNA-Mediated Signaling by Proteins with 4Fe−4S Clusters Is Necessary for Genomic Integrity

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

DNA electrochemistry of the E. coli helicase, DinG

Coli helicase DinG, which can be induced by DNA damage, previously has been shown to have a [4Fe-4S] cluster. While the primary in vivo role of DinG has not been revealed, the protein has been implicated in clearing stalled replication forks that arise from the collision of oppositely oriented transcription and replication machinery. We have explored the DNA-bound redox potential of DinG using electrochem. on gold modified with a helicase substrate, a 20-mer oligonucleotide with a 15-mer overhang. The [4Fe-4S] cluster can be reduced and oxidized reversibly at a DNA-bound redn. potential of ∼ 80 mV vs. This DNA-mediated electrochem. signal, moreover, is stimulated by ATP-hydrolysis. Results from cellular activity assays suggest that DinG may cooperate with other DNA-processing enzymes that have [4Fe-4S] clusters to locate and process DNA damage products

DNA-mediated signaling by the E. coli helicase, DinG

The protein DinG is an ATP-dependent helicase from E. coli that contains a 4Fe-4S cluster. DNA-modified gold electrodes were used to measure the midpoint redox potential of the DNA-bound protein, which was found to be ∼80 mV vs. Using a 20-mer with a 15-mer single-stranded overhang as a helicase substrate on the DNA-modified electrodes, it was shown that enzymic activity via the hydrolysis of ATP increased the intensity of the electrochem. signal intensity. Whether DinG and EndoIII, a base excision repair enzyme also contg. a 4Fe- 4S cluster, use DNA-mediated charge transport (CT) chem. for inter-protein signaling was tested using several techniques. Using a single mol. at. force microscopy assay, it was shown that DinG and EndoIII preferentially redistribute to strands of DNA that contain DNA damage via long-range DNA-mediated CT. To test this signaling within cells, genetics expts. were used that strongly suggest that DinG and EndoIII utilize DNA-mediated signaling to cooperate in redistributing DinG to its target lesion

DNA-Mediated Redox Signaling in Bacterial Nucleotide Excision Repair by UvrC

Our laboratory has proposed redox signaling among DNA repair proteins containing 4Fe-4S clusters through DNA charge transport (DNA CT) as a first step in lesion detection. Recently, we have explored whether UvrC, the endonuclease in Nucleotide Excision Repair (NER), may also contain a 4Fe-4S cluster. A new purification route was developed to express an MBP-UvrC fusion protein, and evidence of a 4Fe-4S cluster was seen by UV-Visible (UV-Vis) and Electron Paramagnetic Resonance (EPR) spectroscopy. Like other 4Fe-4S repair proteins studied in our lab, MBP-UvrC can participate in DNA CT chemistry, as evidenced by its electrochemical activity on DNA-modified gold electrodes. Complementing in vivo genetic assays have been developed and indicate that DNA-mediated signaling between UvrC and other DNA-processing enzymes containing 4Fe-4S clusters is occurring. Taken together, these results have suggested that UvrC is part of a network of 4Fe-4S proteins that communicate using DNA CT to find lesions and maintain genomic integrity. Additional in vitro and in vivo characterization is underway to understand further the biological implications of the newly-discovered, DNA-mediated redox chemistry of UvrC

DNA-mediated redox signaling by UvrC

The E. coli UvrC protein is an integral part of the DNA nucleotide excision repair pathway. Our lab. has focused on studies of redox DNA signaling among DNA repair proteins contg. 4Fe- 4S clusters. We have now explored whether UvrC may also contain a 4Fe- 4S cluster. A new purifn. route was developed to express an MBP- UvrC fusion. The purified protein shows a UVvisible absorbance spectrum consistent with that of a 4Fe- 4S cluster. ESR expts. of reduced UvrC give rise to a signal indicative of a 4Fe- 4S cluster. Electrochem. expts. indicate that the protein is electrochem. active on DNA- modified gold electrodes. Finally, genetic methods to study possible DNA- mediated signaling between UvrC and other DNA- processing enzymes contg. 4Fe- 4S clusters are being examd