MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.

Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial-mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.targeted therapy | gene therapy P ancreatic cancer is an aggressive disease that develops in a relatively symptom-free manner and in most cases, is already advanced at the time of diagnosis (1). It has one of the highest fatality rates of all cancers and is one of the leading causes of cancer-related deaths in the Western world (1, 2). Pancreatic ductal adenocarcinoma (PDA) is the most common pancreatic neoplasm, responsible for 95% of pancreatic cancer cases (3). Genetic alterations in the KRAS signaling pathway are involved in over 90% of pancreatic cancer cases (4-6). KRAS mutations were shown to be an early event in the development of pancreatic cancer (5,7,8).The most common KRAS mutation of the human pancreas adenocarcinoma is a gain-of-function substitution mutation of glycine at codon 12 to aspartate (G12D) (5, 9-11). Moreover, PDA cancer cell growth was shown to be dependent on the activity of the mutated KRAS (5, 11) and accordingly, silencing KRAS has proven effective in controlling pancreatic cell line proliferation (12). Here, we aimed to harness the advantages of siRNA technology as a therapeutic modality for pancreatic cancer.Parenteral controlled drug delivery systems are used to improve and advance the therapeutic effects of drug treatments by providing optimized local drug concentrations over prolonged periods of time, reduction of side effects, and cost reduction (13). A prominent method of controlling the release rate of a drug in a pharmaceutical dosage is to embed the active ag...

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence-and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression. malaria | Plasmodium falciparum | var genes | noncoding RNA | exclusive expression

DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash͞ quench technique, is found to promote oxidation of the 1؉ clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5-G of a 5-GG-3 doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With rutheniumtethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair.electron transfer ͉ iron-sulfur cluster ͉ oxidative DNA damage D NA-mediated charge transport (CT) from a distance to generate oxidative damage was first demonstrated in an assembly containing a tethered metallointercalator (1). In this assembly, photoinduced oxidative damage of the 5Ј-G of 5Ј-GG-3Ј sites was observed; this damage pattern has since become the hallmark of DNA CT chemistry, and long-range oxidative damage has been confirmed by using a variety of pendant oxidants (2-6). Long-range oxidative DNA damage has been demonstrated over a distance of at least 200 Å (7, 8). Indeed, DNA either packaged in nucleosome core particles (9) or inside the cell nucleus (10) has been found to be susceptible to long-range oxidative damage. Chemically well defined assemblies, consisting of DNA duplexes with covalently bound oxidants, have been particularly useful in establishing the sensitivity of DNA CT to base-stacking perturbation (11-16). Recently, analogous studies probing long-range reductive chemistry on DNA has been probed both in solution (17-20) and on DNAmodified surfaces (14,15,21). As with oxidation chemistry, these reactions show only small variations in rate with distance but are remarkably sensitive to perturbations in the intervening base pair stack. Mechanistic descriptions for DNA CT focused first on a mixture of hopping and tunneling. A phonon-assisted polaron model has also been put forth (22). Studies as a function of temperature have shown the CT process to be gated by base pair dynamics; in fact, base pair motions are required for CT (23, 24).We have therefore described DNA CT in the context of transport among delocalized DNA domains formed and dissolved based on sequence-dependent DNA dynamics.Given the exquisite sensitivity of DNA CT to DNA lesions and mismatches, we have recently explored a possible role for DNA CT in repair. We demonstrated that redox activity required DNA binding for MutY (25), a base excision repair (BER) enzyme fr...

The electrochemistry of DNA films modified with different redox probes linked to DNA through saturated and conjugated tethers was investigated. Experiments feature two redox probes bound to DNA on two surfaces:  anthraquinone (AQ)-modified uridines incorporated into thiolated DNA on gold (Au) and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-modified uridines in pyrene-labeled DNA on highly oriented pyrolytic graphite (HOPG). The electrochemistry of these labels when incorporated into DNA has been examined in DNA films containing both well matched and mismatched DNA. DNA-mediated electrochemistry is found to be effective for the TEMPO probe linked with an acetylene linker but not for a saturated TEMPO connected through an ethylenediamine linker. For the AQ probe, DNA-mediated electrochemistry is found with an acetylene linker to uridine but not with an alkyl chain to the 5‘ terminus of the oligonucleotide. Large electrochemical signals and effective discrimination of intervening base mismatches are achieved for the probes connected through the acetylene linkages, while probes connected through saturated linkages exhibit small electrochemical signals associated only with direct surface to probe charge transfer and poor mismatch discrimination. Thus DNA electrochemistry with these probes is dramatically influenced by the chemical nature of their linkage to DNA. These results highlight the importance of effective coupling into the π-stack for long-range DNA-mediated electrochemistry.

Detection of mRNA alterations is a promising approach for identifying biomarkers as means of differentiating benign from malignant lesions. By choosing the KRAS oncogene as a target gene, two types of molecular beacons (MBs) based on either phosphothioated DNA (PS-DNA-MB) or peptide nucleic acid (TO-PNA-MB, where TO = thiazole orange) were synthesized and compared in vitro and in vivo. Their specificity was examined in wild-type KRAS (HT29) or codon 12 point mutation (Panc-1, SW480) cells. Incubation of both beacons with total RNA extracted from the Panc-1 cell line (fully complementary sequence) showed a fluorescent signal for both beacons. Major differences were observed, however, for single mismatch mRNA transcripts in cell lines HT29 and SW480. PS-DNA-MB weakly discriminated such single mismatches in comparison to TO-PNA-MB, which was profoundly more sensitive. Cell transfection of TO-PNA-MB with the aid of PEI resulted in fluorescence in cells expressing the fully complementary RNA transcript (Panc-1) but undetectable fluorescence in cells expressing the K-ras mRNA that has a single mismatch to the designed TO-PNA-MB (HT29). A weaker fluorescent signal was also detected in SW480 cells; however, these cells express approximately one-fifth of the target mRNA of the designed TO-PNA-MB. In contrast, PS-DNA-MB showed no fluorescence in all cell lines tested post PEI transfection. Based on the fast hybridization kinetics and on the single mismatch discrimination found for TO-PNA-MB we believe that such molecular beacons are promising for in vivo real-time imaging of endogenous mRNA with single nucleotide polymorphism (SNP) resolution.