Showing papers by "Jacqueline K. Barton published in 2021"

DNA Electrochemistry: Charge-Transport Pathways through DNA Films on Gold.

Abstract: 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.

Rhodium Complexes Targeting DNA Mismatches as a Basis for New Therapeutics in Cancers Deficient in Mismatch Repair.

Abstract: 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.

The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling.

Abstract: Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase e (Pol e) 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, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo-, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo- 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 Pol2COREexo-, 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 Pol2COREexo- bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo-. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo- via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol e.