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

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 Protection by the Bacterial Ferritin Dps via DNA Charge Transport

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. Here we examine the mechanism of E. coli Dps protection of DNA, specifically whether this DNA-binding protein can utilize DNA charge transport through the base pair π-stack to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate DNA damage localized to guanine repeats, the sites of lowest potential in DNA. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps, significantly attenuates the yield of oxidative DNA damage. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA charge transport may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance

Luminescence of [Ru(bpy)_2(dppz)]^(2+) Bound to RNA Mismatches

The luminescence of rac-[Ru(bpy)_2(dppz)]^(2+) (bpy = 2,2′-bipyridine and dppz = dipyrido[3,2-a:2′,3′-c]phenazine) was explored in the presence of RNA oligonucleotides containing a single RNA mismatch (CA and GG) in order to develop a probe for RNA mismatches. While there is minimal luminescence of [Ru(bpy)_2(dppz)]^(2+) in the presence of matched RNA due to weak binding, the luminescence is significantly enhanced in the presence of a single CA mismatch. The luminescence differential between CA mismatched and matched RNA is substantially higher compared to the DNA analogue, and therefore, [Ru(bpy)_2(dppz)]^(2+) appears to be also a sensitive light switch probe for a CA mismatch in duplex RNA. Although the luminescence intensity is lower in the presence of RNA than DNA, Förster resonance energy transfer (FRET) between the donor ruthenium complex and FRET acceptor SYTO 61 is successfully exploited to amplify the luminescence in the presence of the mismatch. Luminescence and quenching studies with sodium iodide suggest that [Ru(bpy)_2(dppz)]^(2+) binds to these mismatches via metalloinsertion from the minor groove. This work provides further evidence that metalloinsertion is a general binding mode of octahedral metal complexes to thermodynamically destabilized mismatches not only in DNA but also in RNA

A Ruthenium(II) Complex as a Luminescent Probe for DNA Mismatches and Abasic Sites

[Ru(bpy)_2(BNIQ)]^(2+) (BNIQ = Benzo[c][1,7]naphthyridine-1-isoquinoline), which incorporates the sterically expansive BNIQ ligand, is a highly selective luminescent probe for DNA mismatches and abasic sites, possessing a 500-fold higher binding affinity toward these destabilized regions relative to well-matched base pairs. As a result of this higher binding affinity, the complex exhibits an enhanced steady-state emission in the presence of DNA duplexes containing a single base mismatch or abasic site compared to fully well-matched DNA. Luminescence quenching experiments with Cu(phen)_2^(2+) and [Fe(CN)_6]^(3–) implicate binding of the complex to a mismatch from the minor groove via metalloinsertion. The emission response of the complex to different single base mismatches, binding preferentially to the more destabilized mismatches, is also consistent with binding by metalloinsertion. This work shows that high selectivity toward destabilized regions in duplex DNA can be achieved through the rational design of a complex with a sterically expansive aromatic ligand

Sulfur K-Edge XAS Studies of the Effect of DNA Binding on the [Fe_4S_4] Site in EndoIII and MutY

S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe_4S_4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe–S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron–thiolate and iron–sulfide bonds would stabilize the oxidized state of the [Fe_4S_4] clusters. The results are compared to those on previously studied [Fe_4S_4] model complexes, ferredoxin (Fd), and to new data on high-potential iron–sulfur protein (HiPIP). A limited decrease in covalency is observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe_4S_4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron–sulfur bonds. In EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior

Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA

Escherichia coli endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S] clusters and that serve to maintain the integrity of the genome after oxidative stress. Electrochemical studies on highly oriented pyrolytic graphite (HOPG) revealed that DNA binding by EndoIII leads to a large negative shift in the midpoint potential of the cluster, consistent with stabilization of the oxidized [4Fe4S]^(3+) form. However, the smooth, hydrophobic HOPG surface is nonideal for working with proteins in the absence of DNA. In this work, we use thin film voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the cluster, all show DNA-free potentials within error of wild type; similarly, the presence of negatively charged poly-L-glutamate does not lead to a significant potential shift. Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster, helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have similar DNA-bound potentials

State of the art: refinement of multiple sequence alignments

BACKGROUND: Accurate multiple sequence alignments of proteins are very important in computational biology today. Despite the numerous efforts made in this field, all alignment strategies have certain shortcomings resulting in alignments that are not always correct. Refinement of existing alignment can prove to be an intelligent choice considering the increasing importance of high quality alignments in large scale high-throughput analysis. RESULTS: We provide an extensive comparison of the performance of the alignment refinement algorithms. The accuracy and efficiency of the refinement programs are compared using the 3D structure-based alignments in the BAliBASE benchmark database as well as manually curated high quality alignments from Conserved Domain Database (CDD). CONCLUSION: Comparison of performance for refined alignments revealed that despite the absence of dramatic improvements, our refinement method, REFINER, which uses conserved regions as constraints performs better in improving the alignments generated by different alignment algorithms. In most cases REFINER produces a higher-scoring, modestly improved alignment that does not deteriorate the well-conserved regions of the original alignment