Electrically monitoring DNA repair by photolyase

Abstract

Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the -stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV͞blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA.DNA charge transport ͉ DNA electrochemistry ͉ thymine dimers D NA-modified gold electrodes have proven to be invaluable tools in both the study and the application of DNAmediated charge transport (CT) chemistry (1-10). In these systems, the supramolecular self-assembly of thiol-modified DNA duplexes on a gold surface yields well defined monolayers that can serve as platforms for electrochemical assays of DNA CT. In these assays, the base pair stack is interrogated through the reduction of an intercalated reporter such as methylene blue (5, 7) or daunomycin (2, 8); the yield of reduced probe provides a measure of the efficiency of DNA CT. A wealth of experimental data has established that DNA CT is exquisitely sensitive to even subtle perturbations in the base pair -stack, including single base mismatches (3,8,9). Exploiting this dramatic effect, DNA-modified surfaces have been successfully used in the electrochemical detection of mismatches and base lesions (3, 9). Protein-DNA interactions can also be investigated by using these surfaces. Perturbations of the DNA base pair stack caused by protein binding through base flipping or kinking are clearly detected by using this methodology (10). Indeed, this technique can provide a sensitive probe of protein-DNA binding and reaction.The remarkable sensitivity of CT efficiency to DNA lesions and mismatches, as well as the observation that DNA-binding proteins can modulate long-range CT (10, 11), has provided an impetus for the evaluation of a physiological role for DNA CT. DNA-modified gold surfaces have recently been used in the study of DNA repair proteins posses...

Detection of DNA π-Stack Lesions Using Scanning Electrochemical Microscopy

“…Both methylene blue and ruthenium hexammine bind to the DNA monolayer, yet while methylene blue is known as a redox active intercalator that shows electrochemical activity only as long as individual duplexes forming a DNA monolayer are completely Watson-Crick base paired (2), the ruthenium hexamine is electrostatically attached to the DNA phosphate groups, thus its electrochemistry is insensitive to the base-stacking perturbations within the DNA film (19,20). In our approach, shown schematically in figure 1, a redox mediator, Fe(CN) 6 4-is first oxidized at the SECM tip microelectrode (Pt disk) resulting in a steady-state current due to the hemispherical diffusion of Fe(CN) 6 4-to the tip (21). Subsequently, the SECM tip is lowered proximal to the surface of the electrochemically polarized Au electrode modified with a monolayer of ds-DNA containing either ruthenium hexammine (Fig.…”

Examining Cooperative Binding of Sox2 on DC5 Regulatory Element Upon Complex Formation with Pax6 Through Excess Electron Transfer Assay

“…This long-range electron transfer is possible within a distance of 10-25Å in biological redox reactions (16). It has been proposed that some repair enzymes use electron transfer from redox cofactors to allow the detection of DNA lesions generated by the oxidation at remote site (17)(18)(19). However, it was also observed that EET can go through without redox cofactors as evidenced in the case of Escherichia coli DNA photolyase that can repair thymine dimers without the aid of redox cofactors (20,21).…”

Electrochemical probe for the monitoring of DNA–protein interactions

“…Self-assembled DNA monolayers (Davis and Higson, 2005;Finot et al, 2003;Gooding et al, 2003;Legay et al, 2005;Love et al, 2005) provide attractive platforms for electrical monitoring of DNA-protein interactions (Anne et al, 2007;Boon et al, 2002;DeRosa et al, 2005;Drummond et al, 2003;Gorodetsky et al, 2006;Hianik et al, 2004Hianik et al, , 2005Li et al, 2004;Wang, 2002). A convenient way to obtain DNA films is the immobilization of thiolated modified DNA strands on a gold surface (Chaki and Vijayamohanan, 2002;Davis and Higson, 2005;Herne and Tarlov, 1997;Legay et al, 2007;Love et al, 2005) due to the strong affinity of sulfur compounds for gold (Meunier-Prest et al, 2010).…”