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DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by DNA primase. Primase contains a redox-active 4Fe-4S cluster in the C-terminal domain of the p58 subunit (p58C). The redox state of this 4Fe-4S cluster can be modulated via the transport of charge through the protein and the DNA substrate (redox switching); changes in the redox state of the cluster alter the ability of p58C to associate with its substrate. The efficiency of redox switching in p58C can be altered by mutating tyrosine residues that bridge the 4Fe-4S cluster and the nucleic acid binding site. Here, we report the effects of mutating bridging tyrosines to phenylalanines in yeast p58C. High-resolution crystal structures show that these mutations, even with six tyrosines simultaneously mutated, do not perturb the three-dimensional structure of the protein. In contrast, measurements of the electrochemical properties on DNA-modified electrodes of p58C containing multiple tyrosine to phenylalanine mutations reveal deficiencies in their ability to engage in DNA charge transport. Significantly, this loss of electrochemical activity correlates with decreased primase activity. While single-site mutants showed modest decreases in activity compared to that of the wild-type primase, the protein containing six mutations exhibited a 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for charge transport in yeast p58C exist, but inhibiting these pathways together diminishes the ability of yeast primase to generate primers. These results support a model in which redox switching is essential for primase activity.

Over the past 25 years, collective evidence has demonstrated that the DNA base-pair stack serves as a medium for charge transport chemistry in solution and on DNA-modified gold surfaces. Since this charge transport depends sensitively upon the integrity of the DNA base pair stack, perturbations in base stacking, as may occur with DNA base mismatches, lesions, and protein binding, interrupt DNA charge transport (DNA CT). This sensitivity has led to the development of powerful DNA electrochemical sensors. Given the utility of DNA electrochemistry for sensing and in response to recent literature, we describe critical protocols and characterizations necessary for performing DNA-mediated electrochemistry. We demonstrate DNA electrochemistry with a fully AT DNA sequence using a thiolated preformed DNA duplex and distinguish this DNA-mediated chemistry from that of electrochemistry of largely single-stranded DNA adsorbed to the surface. We also demonstrate the dependence of DNA CT on a fully stacked duplex. An increase in the percentage of mismatches within the DNA monolayer leads to a linear decrease in current flow for a DNA-bound intercalator, where the reaction is DNA-mediated; in contrast, for ruthenium hexammine, which binds electrostatically to DNA and the redox chemistry is not DNA-mediated, there is no effect on current flow with mismatches. We find that, with DNA as a well hybridized duplex, upon assembly, a DNA-mediated pathway facilitates the electron transfer between a well coupled redox probe and the gold surface. Overall, this report highlights critical points to be emphasized when utilizing DNA electrochemistry and offers explanations and controls for analyzing confounding results.

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

Up to 20% of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate, and DNA mismatches accumulate as part of these cancers. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) is the most potent, selective compound in this class and acts by targeting DNA mismatches, resulting in preferential cytotoxicity to MMR-deficient cancers. To explore further the cellular mechanism of action of RhPPO, we conjugated the metal complex to a fluorescent probe, cyanine 3 (Cy3). RhPPO-Cy3 binds DNA mismatches and retains the selectivity and potent cytotoxic activity of RhPPO for MMR-deficient cell lines. RhPPO-Cy3 forms discrete foci in the cell nucleus that overlap with sites of DNA damage, suggesting that the lesions occur at or near DNA mismatch sites. RhPPO-Cy3 foci persist over time, despite initial processing of the lesion and recruitment of repair proteins, consistent with the idea that the complex binding to a mismatch prevents repair. RhPPO-Cy3 binding does not lead to activation of p53 and the apoptotic pathway. Together, these findings support the idea that RhPPO-Cy3 binding leads to irreversible DNA damage at DNA mismatches that enables selective cytotoxicity to MMR-deficient cells.

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 ∼500-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.

Cancers with microsatellite instability (MSI), which include ≤20% of solid tumors, are characterized by resistance to chemotherapy due to deficiency in the DNA mismatch repair (MMR) pathway. Rhodium metalloinsertors make up a class of compounds that bind DNA mismatches with high specificity and show selective cytotoxicity in MSI cancer cells. We determined that rhodium complexes with an N∧O coordination showed significantly increased cell potency compared with that of N∧N-coordinated compounds, and we identified [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) as the most potent, selective compound in this class. Using matched cell lines that are MMR-deficient (HCT116O) and MMR-proficient (HCT116N), we demonstrated that RhPPO preferentially activates the DNA damage response and inhibits DNA replication and cell proliferation in HCT116O cells, leading to cell death by necrosis. Using a fluorescent conjugate of RhPPO, we established that the metalloinsertor localizes to DNA mismatches in the cell nucleus and causes DNA double-strand breaks at or near the mismatch sites. Evaluation of RhPPO across MMR-deficient and MMR-proficient cell lines confirmed the broad potential for RhPPO to target MSI cancers, with cell potency significantly higher than that of platinum complexes used broadly as chemotherapeutics. Moreover, in a mouse xenograft model of MSI cancer, RhPPO shows promising antitumor activity and increased survival. Thus, our studies indicate that RhPPO is a novel DNA-targeted therapy with improved potency and selectivity over standard-of-care platinum-based chemotherapy and, importantly, that DNA mismatches offer a critical new target in the design of chemotherapeutics for MSI cancers.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2_(CORE), are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2_(CORE)exo–, was used to limit DNA degradation. While no redox signal is apparent for Pol2_(CORE)exo– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2_(CORE)exo–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]²⁺, to oxidized Pol2_(CORE)exo– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIII^(Y82A), a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2_(CORE)exo–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2_(CORE)exo– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

SUMMARYExtracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in select organisms, a widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. How these shuttles catalyze electron transfer within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) inPseudomonas aeruginosabiofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes inP. aeruginosabiofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms.

Significance This study describes our first evaluation in mice of the rhodium complex Rh-PPO, which selectively targets DNA base-pair mismatches and shows cytotoxicity within mismatch repair-deficient cancer cells. Rh-PPO displays notable in vivo anticancer activity and increases mouse survival when administered intraperitoneally. This work marks the development of a promising metal chemotherapeutic and a new strategy for mismatch-repair-deficient cancers., Mismatch repair (MMR) deficiencies are a hallmark of various cancers causing accumulation of DNA mutations and mismatches, which often results in chemotherapy resistance. Metalloinsertor complexes, including [Rh(chrysi)(phen)(PPO)]Cl2 (Rh-PPO), specifically target DNA mismatches and selectively induce cytotoxicity within MMR-deficient cells. Here, we present an in vivo analysis of Rh-PPO, our most potent metalloinsertor. Studies with HCT116 xenograft tumors revealed a 25% reduction in tumor volume and 12% increase in survival with metalloinsertor treatment (1 mg/kg; nine intraperitoneal doses over 20 d). When compared to oxaliplatin, Rh-PPO displays ninefold higher potency at tumor sites. Pharmacokinetic studies revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Additionally, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, pointing to a need for more selective delivery methods. Overall, these data show that Rh-PPO inhibits xenograft tumor growth, supporting the strategy of using Rh-PPO as a chemotherapeutic targeted to MMR-deficient cancers.

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.

We report a bifunctional fluorescent probe that combines a rhodium metalloinsertor with a cyanine dye as the fluorescent reporter. The conjugate shows weak luminescence when free in solution or with well matched DNA but exhibits a significant luminescence increase in the presence of a 27-mer DNA duplex containing a central CC mismatch. DNA photocleavage experiments demonstrate that, upon photoactivation, the conjugate cleaves the DNA backbone specifically near the mismatch site on a 27-mer fragment, consistent with mismatch targeting. Fluorescence titrations with the 27-mer duplex containing the CC mismatch reveal a DNA binding affinity of 3.1 × 10^6 M^(–1), similar to that of other rhodium metalloinsertors. Fluorescence titrations using genomic DNA extracted from various cell lines demonstrate a clear discrimination in fluorescence between those cell lines that are proficient or deficient in mismatch repair. This differential luminescence reflects the sensitive detection of the mismatchrepair-deficient phenotype.

S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe4S4] 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 [Fe4S4] clusters. The results are compared to those on previously studied [Fe4S4] 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 [Fe4S4] 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 ...

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)]^(2+) (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

DNA has been exploited as a biological target of chemotherapeutics since the 1940s. Traditional chemotherapeutics, such as cisplatin and DNA-alkylating agents, rely primarily on increased uptake by rapidly proliferating cancer cells for therapeutic effects, but this strategy can result in off-target toxicity in healthy tissue. Recently, research interests have shifted towards targeted chemotherapeutics, in which a drug targets a specific biological signature of cancer, resulting in selective toxicity towards cancerous cells. Here, we review a family of complexes, termed rhodium metalloinsertors, that selectively target DNA base pair mismatches, a hallmark of mismatch repair (MMR)-deficient cancers. These rhodium metalloinsertors bind DNA mismatches with high specificity and display high selectively in killing MMR-deficient versus MMR-proficient cells. This cell selectivity is unique among small molecules that bind DNA. Current generations of rhodium metalloinsertors have shown nanomolar potency along with high selectivity towards MMR-deficient cells, and show promise as a foundation for a new family of chemotherapeutics for MMR-deficient cancers.

Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)_2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)_2 is substituted with poly(dAdT)_2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress.

Biological electron transfer reactions between metal cofactors are critical to many essential processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge through its π-stacked nitrogenous bases. Increasingly, [4Fe4S] clusters, generally redox-active cofactors, have been found to be associated with enzymes involved in DNA processing. DNA-binding enzymes containing [4Fe4S] clusters can thus utilize DNA charge transport (DNA CT) for redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT signaling may represent the first step in the search for DNA lesions by proteins containing [4Fe4S] clusters that are involved in DNA repair. Here we describe research carried out to examine the chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT among metalloproteins represents powerful chemistry for redox signaling at long range within the cell.

Defects in DNA mismatch repair (MMR) are commonly found in various cancers, especially in colorectal cancers. Despite the high prevalence of MMR‐deficient cancers, mismatch‐targeted therapeutics are limited and diagnostic tools are indirect. Here, we examine the cytotoxic properties of a rhodium metalloinsertor, [Rh(phen)(chrysi)(PPO)]^(2+)(RhPPO) in 27 diverse colorectal cancer cell lines. Despite the low frequency of genomic mismatches and the non‐covalent nature of the RhPPO‐DNA lesion, RhPPO is on average five times more potent than cisplatin. Importantly, the biological target and profile for RhPPO differs from that of cisplatin. A fluorescent metalloinsertor, RhCy3, was used to demonstrate that the cellular target of RhPPO is the DNA mismatch. RhCy3 represents a direct probe for MMR‐deficiency and correlates directly with the cytotoxicity of RhPPOacross different cell lines. Overall, our studies clearly indicate that RhPPO and RhCy3 are promising anticancer and diagnostic probes for MMR‐deficient cancers, respectively.

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., The length over which DNA-mediated charge transport between [4Fe4S] repair proteins is effective is found to be ∼3500 base pairs, providing a route for a rapid genome search for DNA lesions.

Significance Redox switching driven by [4Fe4S] cluster cofactors modulates DNA binding affinity in proteins, providing a rapid, efficient method of substrate binding and dissociation. Our study establishes an essential redox switch with an aromatic pathway through the yeast DNA primase; a single-residue mutation at position 397 along this redox pathway causes [4Fe4S] cluster degradation and is lethal in yeast., 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.

The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C_(272-464) construct used for that study and that of a N-terminally shifted p58C_(266-456) construct and consequently, an assumption that p58C_(272-464) has abnormal physical and functional properties. To address this controversy, a new p58C_(266-464) construct containing all residues was crystallized under the conditions previously used for crystallizing p58C_(272-464), and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C_(266-464) exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C_(272-464). Moreover, in solution, circular dichroism and ^(15)N,^1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C_(266-464) were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C_(272-464). Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.

Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)2 Fe(NO)2]-, and Roussin’s red ester, [(μ-Cys)2Fe2(NO)4], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin’s red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with 14N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin’s red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with 14NO and 15NO support the assignment of a protein-bound [(μ-Cys)2Fe2(NO)4] Roussin’s red ester. The [Fe4S4]2+/3+ redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 ± 6 and −674 ± 8 mV vs NHE, respectively, corresponding to a shift of approximately −800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.

The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH-associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic polyposis and family history of early age colon cancer. In bacterial MutY, the [4Fe4S] cluster is redox active, allowing rapid localization to target lesions by long-range, DNA-mediated signalling. In the current study, using DNA electrochemistry, we determine that wild-type MUTYH is similarly redox-active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to [3Fe4S]+, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the [4Fe4S] cluster.

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 reaction of the DNA bases with radical species generated by radiation, carcinogens, or oxidative stress can lead to mutagenic damage [1]. The efficiency and dynamics of radical transport through the DNA helix therefore hold profound biological implications. Intriguing questions concerning charge migration through DNA arise that can now begin to be addressed through well-defined chemical experiments. Does radical migration through DNA occur over long molecular distances? How is it modulated by DNA sequence and the structural variations in DNA? Is it physiologically important? How general is this phenomenon? These are issues that need to be addressed in the context of delineating mechanisms of DNA damage and repair.

Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base pair mismatches in vitro and kill mismatch repair (MMR)-deficient cells at lower concentrations than their MMR-proficient counterparts. A family of metalloinsertors containing rhodium–oxygen ligand coordination, termed “Rh–O” metalloinsertors, has been prepared and shown to have a significant increase in both overall potency and selectivity toward MMR-deficient cells regardless of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies with the second generation of Rh–O metalloinsertors in which an ancillary ligand is varied in both steric bulk and lipophilicity. These complexes, of the form [Rh(L)(chrysi)(PPO)]^(2+), all include the O-containing PPO ligand (PPO = 2-(pyridine-2-yl)propan-2-ol) and the aromatic inserting ligand chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L is a phenanthroline or bipyridyl derivative. The Rh–O metalloinsertors in this family all show micromolar binding affinities for a 29-mer DNA hairpin containing a single CC mismatch. The complexes display comparable lipophilic tendencies and pK_a values of 8.1–9.1 for dissociation of an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes containing the phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient cells at nanomolar concentrations. Using mass spectral analyses, it is shown that the complexes are taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an off-target organelle that, when targeted by parent metalloinsertors, can lead to nonselective cytotoxicity. Overall, these Rh–O metalloinsertors have distinct and improved behavior compared to previous generations of parent metalloinsertors, making them ideal candidates for further therapeutic assessment.

DNA base pair mismatches occur naturally in cells as a result of incorporation errors and damage. Most cells are able to identify and correct these mistakes before replication, allowing for high genome fidelity between cellular generations. In some forms of cancer, however, proteins involved in the machinery of mismatch repair (MMR) undergo mutation, making those cells unable to correct mismatches and leading to an increase in mutations. Since higher mismatch frequency serves as an early indicator of cancer progression, for many researchers mismatches have provided a novel target for the design of organic and inorganic small-molecule therapeutics. In particular, transition metal complexes have shown great promise in this context owing to their valuable spectroscopic and photophysical properties and flexibility with respect to modification of their coordination spheres. Thus far, experimental designs have ranged from targeting the thermodynamic destabilization of mismatched sites to the hydrogen-bonding pattern of specific mismatched base pairs. Here, we review the diversity, practical application, and evolution of mismatch-targeting small molecules, with an emphasis on rhodium metalloinsertors and luminescent ruthenium compounds. Importantly, we highlight the discovery of metalloinsertion, a noncovalent DNA binding mode that is specific towards destabilized sites, such as mismatches, within the DNA duplex.

How birds sense the variations in Earth’s magnetic field for navigation is poorly understood, although cryptochromes, proteins homologous to photolyases, have been proposed to participate in this magnetic sensing. Here, in electrochemical studies with an applied magnetic field, we monitor the repair of cyclobutane pyrimidine dimer lesions in duplex DNA by photolyase, mutants of photolyase, and a modified cryptochrome. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 gauss. This high sensitivity to weak magnetic fields depends upon a fast radical pair reaction on the thymines leading to repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be used in a biological compass informed by variations in Earth’s magnetic field., DNA electrochemistry studies illustrate how thymine dimer repair by photolyase and a truncated cryptochrome depends upon weak magnetic fields and thus may serve as a biological compass.

The Priestley Address of 2015 Priestley Medalist Jacqueline K. Barton, given at the 249th ACS National Meeting, Denver, March 24, 2015. First let me say how very honored I am to be receiving this award. I never imagined I would be in this position. And the American Chemical Society, the chemistry community, has already given me so very much. So thank you from the bottom of my heart. Frequently when I travel to different chemistry departments, I end up talking with young people, and they ask about my career path. In fact, just before hearing about this award, I was at Penn State, and at the poster session, a young woman asked very straightforwardly, “So how did you get to be you?” Let’s be clear, it wasn’t by design. It was the result of a succession of accidents and opportunities. So I thought that would be the basis of my ...

Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were ^(13)C_2D_2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward ^(13)C_2D_2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

A [4Fe4S]^(2+) cluster in the C-terminal domain of the catalytic subunit of the eukaryotic B-family DNA polymerases is essential for the formation of active multi-subunit complexes. Here we use a combination of electrochemical and biochemical methods to assess the redox activity of the [4Fe4S]^(2+) cluster in Saccharomyces cerevisiae polymerase (Pol) δ, the lagging strand DNA polymerase. We find that Pol δ bound to DNA is indeed redox-active at physiological potentials, generating a DNA-mediated signal electrochemically with a midpoint potential of 113 ± 5 mV versus NHE. Moreover, biochemical assays following electrochemical oxidation of Pol δ reveal a significant slowing of DNA synthesis that can be fully reversed by reduction of the oxidized form. A similar result is apparent with photooxidation using a DNA-tethered anthraquinone. These results demonstrate that the [4Fe4S] cluster in Pol δ can act as a redox switch for activity, and we propose that this switch can provide a rapid and reversible way to respond to replication stress.

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.

Baranovskiy et al . and Pellegrini argue that, based on structural data, the path for charge transfer through the [4Fe4S] domain of primase is not feasible. Our manuscript presents electrochemical data directly showing charge transport through DNA to the [4Fe4S] cluster of a primase p58C construct and a reversible switch in the DNA-bound signal with oxidation/reduction, which is inhibited by mutation of three tyrosine residues. Although the dispositions of tyrosines differ in different constructs, all are within range for microsecond electron transfer.

[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)22+ 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., The luminescent complex [Ru(bpy)2(BNIQ)]2+ selectivity targets mismatched and abasic sites in duplex DNA and exhibits an enhanced emission intensity in the presence of these defect sites relative to well-matched base pairs.

The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH­associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic polyposis and family history of early­age colon cancer. In bacterial MutY, the [4Fe4S] cluster is redox active, allowing rapid localization to target lesions by long­range, DNA­mediated signalling. In the current study, using DNA electrochemistry, we determine that wild­type MUTYH is similarly redox­active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to [3Fe4S]+, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the [4Fe4S] cluster.

A DNA electrochemistry platform has been developed to probe proteins bound to DNA electrically. Here gold electrodes are modified with thiol-modified DNA, and DNA charge transport chemistry is used to probe DNA binding and enzymatic reaction both with redox-silent and redox-active proteins. For redox-active proteins, the electrochemistry permits the determination of redox potentials in the DNA-bound form, where comparisons to DNA-free potentials can be made using graphite electrodes without DNA modification. Importantly, electrochemistry on the DNA-modified electrodes facilitates reaction under aqueous, physiological conditions with a sensitive electrical measurement of binding and activity.

Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology.

Strategies to detect human DNA methyltransferases are needed, given that aberrant methylation by these enzymes is associated with cancer initiation and progression. Here we describe a non-radioactive, antibody-free, electrochemical assay in which methyltransferase activity on DNA-modified electrodes confers protection from restriction for signal-on detection. We implement this assay with a multiplexed chip platform and show robust detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Essential to work with human methyltransferases, our unique assay design allows activity measurements on both unmethylated and hemimethylated DNA substrates. We validate this assay by comparison with a conventional radioactive method. The advantages of electrochemistry over radioactivity and fluorescence make this assay an accessible and promising new approach for the sensitive, label-free detection of human methyltransferase activity.

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, Forster 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.

DNA charged with regulating replication DNA can transport electrical charge over long distances and has the potential to act as a signaling system. The iron-sulfur complex [4Fe4S] found in some proteins is known to be involved in redox reactions. The eukaryotic DNA primase is involved in DNA replication and contains a [4Fe4S] cluster that is required for its RNA primer synthesis activity. O'Brien et al. show that the [4Fe4S] cluster in DNA primase can regulate the protein's DNA binding activity through DNA-mediated charge transfer. This in turn plays a role in primer initiation and length determination. Science , this issue p. eaag1789

Recent work suggests that electrons can travel through DNA and other chiral molecules in a spin-selective manner, but little is known about the origin of this spin selectivity. Here we describe experiments on magnetized DNA-modified electrodes to explore spin-selective electron transport through hydrated duplex DNA. Our results show that the two spins migrate through duplex DNA with a different yield and that spin selectivity requires charge transport through the DNA duplex. Significantly, shifting the same duplex DNA between right-handed B- and left-handed Z-forms leads to a diode-like switch in spin selectivity; which spin moves more efficiently through the duplex depends upon the DNA helicity. With DNA, the supramolecular organization of chiral moieties, rather than the chirality of the individual monomers, determines the selectivity in spin, and thus a conformational change can switch the spin selectivity.

[Ru(Me_4phen)_2dppz]^(2+) serves as a luminescent “light switch” for single base mismatches in DNA. The preferential luminescence enhancement observed with mismatches results from two factors: (i) the complex possesses a 26-fold higher binding affinity toward the mismatch compared to well-matched base pairs, and (ii) the excited state emission lifetime of the ruthenium bound to the DNA mismatch is 160 ns versus 35 ns when bound to a matched site. Results indicate that the complex binds to the mismatch through a metalloinsertion binding mode. Cu(phen)_2^(2+) quenching experiments show that the complex binds to the mismatch from the minor groove, characteristic of metalloinsertion. Additionally, the luminescence intensity of the complex with DNA containing single base mismatches correlates with the thermodynamic destabilization of the mismatch, also consistent with binding through metalloinsertion. This complex represents a potentially new early cancer diagnostic for detecting deficiencies in mismatch repair.