RESUMO
Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a â¼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
Assuntos
DNA Glicosilases/química , DNA Helicases/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Endonucleases/química , Genoma , Proteínas Ferro-Enxofre/química , Animais , Bactérias/genética , Bactérias/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Dano ao DNA , DNA Glicosilases/metabolismo , DNA Glicosilases/ultraestrutura , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/ultraestrutura , Espectroscopia de Ressonância de Spin Eletrônica , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/ultraestrutura , Oxirredução , Ligação Proteica , Transdução de Sinais , TermodinâmicaRESUMO
Understanding of DNA-mediated charge transport (CT) is significant for exploring circuits at the molecular scale. However, the fabrication of robust DNA wires remains challenging due to the persistence length and natural flexibility of DNA molecules. Moreover, CT regulation in DNA wires often relies on predesigned sequences, which limit their application and scalability. Here, we addressed these issues by preparing self-assembled DNA nanowires with lengths of 30-120 nm using structural DNA nanotechnology. We employed these nanowires to plug individual gold nanoparticles into a circuit and measured the transport current in nanowires with an optical imaging technique. Contrary to the reported cases with shallow or no length dependence, a fair current attenuation was observed with increasing nanowire length, which experimentally confirmed the prediction of the incoherent hopping model. We also reported a mechanism for the reversible CT regulation in DNA nanowires, which involves dynamic transitions in the steric conformation.
Assuntos
Nanopartículas Metálicas , Nanofios , Nanofios/química , Ouro/química , Nanotecnologia/métodos , DNA/químicaRESUMO
Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.
Assuntos
DNA Primase/química , Proteínas Ferro-Enxofre/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Cristalografia por Raios X , DNA Primase/genética , Transporte de Elétrons , Proteínas Ferro-Enxofre/genética , Modelos Moleculares , Mutação , Oxirredução , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA as a medium for electron transfer has been widely used in photolytic processes but is seldom applied to dark reaction of CO2 reduction. A G-quadruplex nanowire (tsGQwire) assembled by guanine tetranucleotides was used to host several metal complexes and further to mediate electron transfer processes in the electrochemical reduction of CO2 catalyzed by these complexes. The tsGQwire modified electrode increased the Faradaic efficiency of cobalt(II) phthalocyanine (CoII Pc) 2.5-folds for CO production than bare CoII Pc electrode, with a total current density of 11.5â mA cm-2 . Comparable Faradaic efficiency of HCOOH production was achieved on tsGQwire electrode when the catalytic center was switched to a GQ targeting Ru complex. The high efficiency and selectivity of electrocatalytic CO2 reduction was attributed to the unique binding of metal complexes on G-quadruplex and electron transfer mediated by GQ nanowire to achieve efficient redox cycling of catalytic centers on the electrode.
RESUMO
The phenomenon of DNA hole transport (HT) has attracted of scientists for several decades, mainly due to its potential application in molecular electronics. As electron holes mostly localize on purine bases in DNA, the majority of scientific effort has been invested into chemically modifying the structures of adenine and guanine in order to increase their HT-mediating properties. In this work we examine an alternative, never yet explored, way of affecting the HT efficiency by forcing electron holes to localize on pyrimidine bases and move between them. Using an enhanced and revised version of our previously developed QM/MM model, we perform simulations of HT through polyadenine, polycytosine, polyguanine, and polythymine stacks according to a multistep hopping mechanism. From these simulations, kinetic parameters for HT are obtained. The results indicate a particularly high efficiency of cytosineâcytosine hopping, which is about ten times higher than the G â G hopping. We also discuss possible improvement of cytosine HT by modifying the oxidoreductive properties of complementary guanine residues.
Assuntos
DNA/química , Elétrons , Modelos Moleculares , Modelos Teóricos , Pirimidinas/química , Algoritmos , Pareamento de Bases , Sequência de Bases , Transporte de Elétrons , Conformação de Ácido Nucleico , Teoria QuânticaRESUMO
Replication of DNA in eukaryotes is primarily executed by the combined action of processive DNA polymerases δ and É. These enzymes cannot initiate synthesis of new DNA without the presence of a primer on the template ssDNA. The primers on both the leading and lagging strands are generated by DNA polymerase α-primase (pol-prim). DNA primase is a DNA-dependent RNA polymerase that synthesizes the first ~10 nucleotides and then transfers the substrate to polymerase α to complete primer synthesis. The mechanisms governing the coordination and handoff between primase and polymerase α are largely unknown. Isolated DNA primase contains a [4Fe-4S]2+ cluster that has been shown to serve as a redox switch modulating DNA binding affinity. This discovery suggests a mechanism for modulating the priming activity of primase and handoff to polymerase α. In this chapter, we briefly discuss the current state of knowledge of primase structure and function, including the role of its iron-sulfur cluster. This is followed by providing the methods for expressing, purifying, and biophysically/structurally characterizing primase and its iron-sulfur cluster-containing domain, p58C.