Sensing DNA through DNA Charge Transport

DNA charge transport chemistry involves the migration of charge over long molecular distances through the aromatic base pair stack within the DNA helix. This migration depends upon the intimate coupling of bases stacked one with another, and hence any perturbation in that stacking, through base modifications or protein binding, can be sensed electrically. In this review, we describe the many ways DNA charge transport chemistry has been utilized to sense changes in DNA, including the presence of lesions, mismatches, DNA-binding proteins, protein activity, and even reactions under weak magnetic fields. Charge transport chemistry is remarkable in its ability to sense the integrity of DNA

The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins

A central question important to understanding DNA repair is how certain proteins are able to search for, detect, and fix DNA damage on a biologically relevant time scale. A feature of many base excision repair proteins is that they contain [4Fe4S] clusters that may aid their search for lesions. In this paper, we establish the importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process. We utilize DNA-modified electrodes to generate repair proteins with [4Fe4S] clusters in the 2+ and 3+ states by bulk electrolysis under an O_2-free atmosphere. Anaerobic microscale thermophoresis results indicate that proteins carrying [4Fe4S]^(3+) clusters bind to DNA 550 times more tightly than those with [4Fe4S]^(2+) clusters. The measured increase in DNA-binding affinity matches the calculated affinity change associated with the redox potential shift observed for [4Fe4S] cluster proteins upon binding to DNA. We further devise an electrostatic model that shows this change in DNA-binding affinity of these proteins can be fully explained by the differences in electrostatic interactions between DNA and the [4Fe4S] cluster in the reduced versus oxidized state. We then utilize atomic force microscopy (AFM) to demonstrate that the redox state of the [4Fe4S] clusters regulates the ability of two DNA repair proteins, Endonuclease III and DinG, to bind preferentially to DNA duplexes containing a single site of DNA damage (here a base mismatch) which inhibits DNA charge transport. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process

Effective Distance for DNA-Mediated Charge Transport between Repair Proteins

The stacked aromatic base pairs within the DNA double helix facilitate charge transport down its length in the absence of lesions, mismatches, and other stacking perturbations. DNA repair proteins containing [4Fe4S] clusters can take advantage of DNA charge transport (CT) chemistry to scan the genome for mistakes more efficiently. Here we examine the effective length over which charge can be transported along DNA between these repair proteins. We define the effective CT distance as the length of DNA within which two proteins are able to influence their ensemble affinity to the DNA duplex via CT. Endonuclease III, a DNA repair glycosylase containing a [4Fe4S] cluster, was incubated with DNA duplexes of different lengths (1.5–9 kb), and atomic force microscopy was used to quantify the binding of proteins to these duplexes to determine how the relative protein affinity changes with increasing DNA length. A sharp change in binding slope is observed at 3509 base pairs, or about 1.2 μm, that supports the existence of two regimes for protein binding, one within the range for DNA CT, one outside of the range for CT; DNA CT between the redox proteins bound to DNA effectively decreases the ensemble binding affinity of oxidized and reduced proteins to DNA. Utilizing an Endonuclease III mutant Y82A, which is defective in carrying out DNA CT, shows only one regime for protein binding. Decreasing the temperature to 4 °C or including metallointercalators on the duplex, both of which should enhance base stacking and decrease DNA floppiness, leads to extending the effective length for DNA charge transport to ∼5300 bp or 1.8 μm. These results thus support DNA charge transport between repair proteins over kilobase distances. The results furthermore highlight the ability of DNA repair proteins to search the genome quickly and efficiently using DNA charge transport chemistry

Sensing DNA through DNA Charge Transport

DNA charge transport chemistry involves the migration of charge over long molecular distances through the aromatic base pair stack within the DNA helix. This migration depends upon the intimate coupling of bases stacked one with another, and hence any perturbation in that stacking, through base modifications or protein binding, can be sensed electrically. In this review, we describe the many ways DNA charge transport chemistry has been utilized to sense changes in DNA, including the presence of lesions, mismatches, DNA-binding proteins, protein activity, and even reactions under weak magnetic fields. Charge transport chemistry is remarkable in its ability to sense the integrity of DNA

Effective Distance for DNA-Mediated Charge Transport between Repair Proteins

The stacked aromatic base pairs within the DNA double helix facilitate charge transport down its length in the absence of lesions, mismatches, and other stacking perturbations. DNA repair proteins containing [4Fe4S] clusters can take advantage of DNA charge transport (CT) chemistry to scan the genome for mistakes more efficiently. Here we examine the effective length over which charge can be transported along DNA between these repair proteins. We define the effective CT distance as the length of DNA within which two proteins are able to influence their ensemble affinity to the DNA duplex via CT. Endonuclease III, a DNA repair glycosylase containing a [4Fe4S] cluster, was incubated with DNA duplexes of different lengths (1.5–9 kb), and atomic force microscopy was used to quantify the binding of proteins to these duplexes to determine how the relative protein affinity changes with increasing DNA length. A sharp change in binding slope is observed at 3509 base pairs, or about 1.2 μm, that supports the existence of two regimes for protein binding, one within the range for DNA CT, one outside of the range for CT; DNA CT between the redox proteins bound to DNA effectively decreases the ensemble binding affinity of oxidized and reduced proteins to DNA. Utilizing an Endonuclease III mutant Y82A, which is defective in carrying out DNA CT, shows only one regime for protein binding. Decreasing the temperature to 4 °C or including metallointercalators on the duplex, both of which should enhance base stacking and decrease DNA floppiness, leads to extending the effective length for DNA charge transport to ∼5300 bp or 1.8 μm. These results thus support DNA charge transport between repair proteins over kilobase distances. The results furthermore highlight the ability of DNA repair proteins to search the genome quickly and efficiently using DNA charge transport chemistry

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm

DNA-processing repair proteins containing redox-active [4Fe4S] metallocofactors facilitate DNA lesion detection

DNA stores vital genetic information from which the foundation of life is built upon. Oxidative stress poses a recurrent threat to genome integrity and a continual risk of cancer development. The ability for charge to be transferred through DNA has implications in its structural maintenance assisted by repair proteins carrying redox-active [4Fe4S] clusters. In this presentation, I will present our work on establishing the importance of the redox state of the [4Fe4S] inorg. cofactor in the DNA damage detection process. Specifically, [4Fe4S] clusters primarily in the oxidized state abolish the ability of repair proteins to differentiate between well-matched and damaged DNA strands. Conversely, [4Fe4S] metallocofactors primarily in the reduced state enable repair proteins to redistribute onto DNA duplexes contg. a C:A mismatch. We further devised a biophys. model that focuses on the electrostatic interactions between [4Fe4S] cluster proteins and DNA to explain the exptl. obsd. change in DNA binding affinity upon switching the oxidn. state of the [4Fe4S] cluster proteins. I will also present our new findings on how far and how fast electrons travel across DNA duplexes. Techniques: (i) DNA-modified electrode, (ii) at.-force-microscopy-based redistribution assay, (iii) gel-based electrophoretic mobility shift assay, (iv) biophys. electrostatic differential binding model, (v) ESR, (vi) UV-visible spectroscopy, (vii) electrochem., (viii) CD. Significance: Repair proteins carrying [4Fe4S] clusters modulate their binding strength to DNA by switching the redox state of the [4Fe4S] inorg. cofactors to regulate the ability of the repair proteins to search for DNA damage via DNA-mediated charge transfer in response to cellular oxidative stress. Figure Captions: (A) Biophys. electrostatic model, (B) DNA-modified electrode to generate primarily reduced or oxidized proteins carrying [4Fe4S] clusters, and (C) DNA damage search mechanism enabled by DNA-mediated electron transport

DNA-processing repair proteins containing redox-active [4Fe4S] metallocofactors facilitate DNA lesion detection

DNA stores vital genetic information from which the foundation of life is built upon. Oxidative stress poses a recurrent threat to genome integrity and a continual risk of cancer development. The ability for charge to be transferred through DNA has implications in its structural maintenance assisted by repair proteins carrying redox-active [4Fe4S] clusters. In this presentation, I will present our work on establishing the importance of the redox state of the [4Fe4S] inorg. cofactor in the DNA damage detection process. Specifically, [4Fe4S] clusters primarily in the oxidized state abolish the ability of repair proteins to differentiate between well-matched and damaged DNA strands. Conversely, [4Fe4S] metallocofactors primarily in the reduced state enable repair proteins to redistribute onto DNA duplexes contg. a C:A mismatch. We further devised a biophys. model that focuses on the electrostatic interactions between [4Fe4S] cluster proteins and DNA to explain the exptl. obsd. change in DNA binding affinity upon switching the oxidn. state of the [4Fe4S] cluster proteins. I will also present our new findings on how far and how fast electrons travel across DNA duplexes. Techniques: (i) DNA-modified electrode, (ii) at.-force-microscopy-based redistribution assay, (iii) gel-based electrophoretic mobility shift assay, (iv) biophys. electrostatic differential binding model, (v) ESR, (vi) UV-visible spectroscopy, (vii) electrochem., (viii) CD. Significance: Repair proteins carrying [4Fe4S] clusters modulate their binding strength to DNA by switching the redox state of the [4Fe4S] inorg. cofactors to regulate the ability of the repair proteins to search for DNA damage via DNA-mediated charge transfer in response to cellular oxidative stress. Figure Captions: (A) Biophys. electrostatic model, (B) DNA-modified electrode to generate primarily reduced or oxidized proteins carrying [4Fe4S] clusters, and (C) DNA damage search mechanism enabled by DNA-mediated electron transport