REDOX CHEMISTRY IN THE GENOME
IRON SULFUR CLUSTERS IN DNA PROCESSING ENZYMES . 3
An unstable ferrous iron from the cofactor can react with hydrogen peroxide in the cellular environment; this Fenton chemistry creates reactive oxygen species that can damage nearby DNA bases [65, 66]. Proteins separated by commas in the Eukarya column are from yeast (left) and humans (right). MutY, however, can denature and revert to the or form, challenging this early conclusion [114].
While the tethered, DNA-bound ruthenium complex luminesced in the absence of the rhodium complex, the luminescence in its presence was quenched by electron transfer occurring through the π-folded dsDNA duplex and remarkably at a distance of > 40 Å [97]. In a subsequent experiment using the 2-aminopurine base analog as the luminescent donor, with guanine serving as the electron acceptor, suppression of electron transfer was also evident, but was attenuated in the presence of a single base mismatch (a lesion that interferes with -stacking), which intervenes between the bound donor and. The aromatic bases in the center of the DNA helix are oriented in such a way that the π-orbitals of the neighboring bases in the duplex overlap with each other.
Indeed, ground-state CT through DNA was observed over 100 base pairs to a bound, intercalating redox probe, Nile blue, using multiplexed DNA-modified gold electrodes, but a single base mismatch in the 100-mer was sufficient to severely attenuate the redox signal [129]. Repair and replication enzymes bearing [4Fe4S] fulfill all these criteria and were thus investigated in the context of DNA CT chemistry, described in the next section.
MEASURING REDOX POTENTIALS OF [4FE4S] ENZYMES
- DNA-Mediated Electrochemistry
- Graphite Electrochemistry to Compare DNA-bound and DNA-
- Spectroscopic Observation of [4Fe4S] Cluster Redox Activ-
- A Shift in Cluster Potential Reflects a Redox Switch in DNA
It was reasonable to consider that binding of the DNA polyanion could change the potential of the group within the protein. Furthermore, the presence of an abasic site on the DNA intervening between the bound protein and the electrode surface served to attenuate the signal from the cluster. Cyclic voltammetry scanning can be used to measure the DNA-mediated redox signaling of CT-proficient proteins (such as WT EndoIII, blue) and CT-deficient mutant proteins (EndoIII Y82A, red).
Highly oriented pyrolytic graphite (HOPG) electrodes were used to measure DNA-free and DNA-bound EndoIII redox potentials [51]. A negative shift of about 200 mV was observed for DNA-bound EndoIII relative to DNA-free EndoIII. This potential shift thermodynamically corresponds to the stabilization of the oxidized state of [4Fe4S]3+ upon binding of the DNA polyanion.
We also demonstrated that protein bound to DNA can be photochemically oxidized, from a distance, using CT of DNA by a distantly cross-linked photooxidant [ 6 ]. We have recently discovered that small-scale thermophoresis can be used under anaerobic conditions to perform DNA binding experiments for [4Fe4S] proteins in the two oxidation states [132].
DNA REPAIR ENZYMES COORDINATING [4FE4S] CENTERS . 12
- Superfamily 2 (SF2) 5 0 → 3 0 Helicases
- Helicase-Nucleases
- Monitoring Redox Signaling Among [4Fe4S] Repair Proteins 16
- Diseases and Cancers Related to Mutations in [4Fe4S] Proteins 19
- DNA Polymerase-α-Primase Begins Replication through
- DNA Polymerases ε and δ Divide Labor between Leading
Additional binding affinity measurements of other repair proteins in reduced and oxidized states are needed to continue to investigate the role of the cluster in the context of each protein. Helicase activity therefore increases the electrochemical signal through better coupling of the group to the π-stacked DNA bases, essentially signaling helicase activity through DNA CT. Two factors have been found to influence the efficiency of the damage search: (i) the CT skill of a protein and (ii) the degree to which the protein population is oxidized [132].
These data underscore that long-range redox signaling between [4Fe4S] enzymes is dependent on the shared DNA-bound redox potential and CT skill of the protein. Moreover, this degradation occurs only with oxidation, which underlines the important role of the group in the performance of redox chemistry. For all the proteins described so far, the [4Fe4S] cluster has been involved in redox chemistry, but not directly in the enzymatic reaction carried out by the protein.
The C-terminal domain of the auxiliary subunit of primase (p58C) and presumably the C-terminal domain of the Polαcatalytic subunit (p180), coordinate [4Fe4S] cofactors. As we saw with repair proteins, the oxidation state of the group regulated DNA binding, providing a redox switch for binding.
MORE [4FE4S] PROTEINS IN DNA PROCESSING
Furthermore, Polδ is stabilized in the presence of stalled forks under replication stress and may play a role in response to changes in the environment [32]. Polδ then remains stalled in the oxidized form, consistent with stalling of Polδ found under conditions of oxidative stress [10]. Several of these proteins have been shown to participate in DNA-mediated redox signaling; characterization of their redox roles is in progress.
Under conditions of oxidative stress, polymerase δ can be converted to the [4Fe4S]3+ state as a means of inhibiting synthesis under poor cellular conditions (53). The translesion polymerase also contains a [4Fe4S] group, coordinated to the catalytic subunit Rev3, which is homologous to other catalytic subunits of family B polymerases. By interacting with PCNA and many other replication factors, the polymerase catalyzes the mutagenic activity of polymerase in the presence of lesions that inhibit replication fork progression (114).
Several of these proteins have been shown to participate in DNA-mediated redox signaling; characterization of their redox roles is ongoing. In interaction with PCNA and many other replication factors, Pol ζ catalyzes mutagenic polymerase activity in the presence of lesions that block replication fork progression [85].
IDENTIFYING NEW [4FE4S] CENTERS IN DNA PROCESSING
Electrochemistry of the [4Fe4S] cluster in base excision repair proteins: tuning the redox potential with DNA. Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY. An iron-sulfur domain of the eukaryotic primase is essential for the synthesis of RNA primers.
Redox control of the DNA damage-inducible protein DinG helicase activity via its iron-sulfur cluster. An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase. Shown is a schematic representation of GIY-YIG endonuclease (30 cuts, light green), cysteine rich (Cys, orange), UvrBC interacting (BC, purple), RNAse H endonuclease (50 cuts, dark green) and helix-turn-helix (( HhH)2teal) domains of UvrC (middle).
A highly conserved aromatic residue (Tyr169) and two conserved proline residues (Pro165 and Pro177) are also located in close proximity to conserved cysteine residues. The percentage of [4Fe4S] cofactor incorporated was calculated by dividing the total [4Fe4S] concentration by the total protein concentration. Gray to suggest that oxygen may be contributing to the degradation of the [4Fe4S] cofactor.
Redox control of DNA damage-induced protein helicase DinG activity through its iron-sulfur cluster. Simple time courses showing the degradation of the [4Fe4S] cofactor in the presence of molecular oxygen after 1 h incubation at 37 °C (again the usual incubation times and temperatures for the activity assay) were performed at protein species concentrations above (µM) those , which is commonly used in the activity assay (nM). Binding to the duplexed 30-mer WM substrate does not slow aerobic degradation of the [4Fe4S] cluster.
Shown is the setup of the Emulsiflex instrument (left) and the regulator for the argon tank (right), which is connected to the Emulsiflex via blue tubing. On the day of purification, the cell pellets were thawed on ice in a glove bag. After applying the lysate to the column, the buffer line was reconnected to the Lysis buffer line.
Noncysteinyl coordination in the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Cysteine mutants of the repair protein MutY and the human homologue, MUTYH, have been well studied in the literature. The work on UvrC until the discovery of the [4Fe4S] cluster was to our knowledge completed under aerobic conditions, presumably with protein apo (and not only diluted protein) [27].
