A synthetic agent ameliorates polycystic kidney disease by promoting apoptosis of cystic cells through increased oxidative stress.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

5-Chloro-2'-deoxycytidine Induces a Distinctive High-Resolution Mutational Spectrum of Transition Mutations In Vivo.

The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C → T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gptΔ C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG → TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA → CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Establishing Linkages Among DNA Damage, Mutagenesis, and Genetic Diseases.

DNA damage by chemicals, radiation, or oxidative stress leads to a mutational spectrum, which is complex because it is determined in part by lesion structure, the DNA sequence context of the lesion, lesion repair kinetics, and the type of cells in which the lesion is replicated. Accumulation of mutations may give rise to genetic diseases such as cancer and therefore understanding the process underlying mutagenesis is of immense importance to preserve human health. Chemical or physical agents that cause cancer often leave their mutational fingerprints, which can be used to back-calculate the molecular events that led to disease. To make a clear link between DNA lesion structure and the mutations a given lesion induces, the field of single-lesion mutagenesis was developed. In the last three decades this area of research has seen much growth in several directions, which we attempt to describe in this Perspective.

Metallocalix[4]arene Polymers for Gravimetric Detection of N-Nitrosodialkylamines.

N-Nitrosamines are found in food, drugs, air, water, and soil. They pose a significant risk to human health because of their carcinogenicity; consequently, materials that can be used to selectively and sensitively detect nitrosamines are needed. In this work, we designed and synthesized two polymers bearing calix[4]arene or 4-tert-butylcalix[4]arene tungsten-imido complexes (PCalixH and PCalixtBu) as N-nitrosodimethylamine (NDMA) receptors. The interaction between metallocalix[4]arene monomers/polymers and NDMA was confirmed by 1H NMR and IR spectroscopy. Single-crystal X-ray analysis further revealed that the host-guest interaction is based on binding of the terminal oxygen of NDMA to tungsten within the calixarene cavity. Gravimetric detection of NDMA was performed on a quartz crystal microbalance (QCM) in air. Both polymers show responses to NDMA, with PCalixtBu exhibiting a low theoretical limit of detection of 5 ppb for NDMA. The sensor also shows high selectivity toward NDMA and moderate humidity tolerance. This work provides a sensitive sensor for detection of NDMA and also offers a class of new, selective, and efficient NDMA receptors for the future design of NDMA sensors and NDMA extraction materials.

DNA repair enzymes ALKBH2, ALKBH3, and AlkB oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine in vitro.

5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is modified to 5hmC, 5fC, and 5caC in vitro by another class of α-KG/Fe(II)-dependent proteins-the DNA repair enzymes in the AlkB family, which include ALKBH2, ALKBH3 in huamn and AlkB in Escherichia coli. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.

Modulation of N-Methyl-N-nitrosourea Mutagenesis in Mouse Embryo Fibroblasts Derived from the gpt Delta Mouse by an Inhibitor of the O6-Methylguanine Methyltransferase, MGMT.

DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is O6-methylguanine (m6G), which base pairs with thymine during replication to cause GC → AT mutations. The gpt delta C57BL/6J mouse strain of Nohmi et al. (Mol. Mutagen 1996, 28, 465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from gpt delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by N-methyl-N-nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental N-nitrosamines, such as N-nitrosodimethylamine and therapeutic agents such as Temozolomide. The DNA repair protein MGMT (O6-methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of Temozolomide in cancer therapy. The gpt delta MEFs were treated with MNU to establish dose-dependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC → AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using O6-methyl-d3-guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation frequency seen in the same cells. This work establishes gpt delta MEFs as a versatile tool for probing mutagenesis by environmental and therapeutic agents and as a cell culture model in which chemical genetics can be used to determine the impact of DNA repair on biological responses to DNA damaging agents.

Mutational spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma.

Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB1, and, as such, is an early detection metric for AFB1-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice.

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2-/- Il10-/- gpt delta mice is influenced by sex.

Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2-/- Il10-/- gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice.

Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity.

Antiviral drugs designed to accelerate viral mutation rates can drive a viral population to extinction in a process called lethal mutagenesis. One such molecule is 5,6-dihydro-5-aza-2'-deoxycytidine (KP1212), a selective mutagen that induces A-to-G and G-to-A mutations in the genome of replicating HIV. The mutagenic property of KP1212 was hypothesized to originate from its amino-imino tautomerism, which would explain its ability to base pair with either G or A. To test the multiple tautomer hypothesis, we used 2D IR spectroscopy, which offers subpicosecond time resolution and structural sensitivity to distinguish among rapidly interconverting tautomers. We identified several KP1212 tautomers and found that >60% of neutral KP1212 is present in the enol-imino form. The abundant proportion of this traditionally rare tautomer offers a compelling structure-based mechanism for pairing with adenine. Additionally, the pKa of KP1212 was measured to be 7.0, meaning a substantial population of KP1212 is protonated at physiological pH. Furthermore, the mutagenicity of KP1212 was found to increase dramatically at pH <7, suggesting a significant biological role for the protonated KP1212 molecules. Overall, our data reveal that the bimodal mutagenic properties of KP1212 result from its unique shape shifting ability that utilizes both tautomerization and protonation.

Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer.

During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused C→T transitions at frequencies ranging from 3-9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase ß replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the C→T transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature C→T mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.

Role of tautomerism in RNA biochemistry.

Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.

1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in Escherichia coli of Various Repair and Replication Backgrounds.

The adverse effects of the human carcinogen 1,3-butadiene (BD) are believed to be mediated by its DNA-reactive metabolites such as 3,4-epoxybut-1-ene (EB) and 1,2,3,4-diepoxybutane (DEB). The specific DNA adducts responsible for toxic and mutagenic effects of BD, however, have yet to be identified. Recent in vitro polymerase bypass studies of BD-induced adenine (BD-dA) adducts show that DEB-induced N6,N6-DHB-dA (DHB = 2,3-dihydroxybutan-1,4-diyl) and 1,N6-γ-HMHP-dA (HMHP = 2-hydroxy-3-hydroxymethylpropan-1,3-diyl) adducts block replicative DNA polymerases but are bypassed by human polymerases η and κ, leading to point mutations and deletions. In contrast, EB-induced N6-HB-dA (HB = 2-hydroxy-3-buten-1-yl) does not block DNA synthesis and is nonmutagenic. In the present study, we employed a newly established in vivo lesion-induced mutagenesis/genotoxicity assay via next-generation sequencing to evaluate the in vivo biological consequences of S-N6-HB-dA, R,R-N6,N6-DHB-dA, S,S-N6,N6-DHB-dA, and R,S-1,N6-γ-HMHP-dA. In addition, the effects of AlkB-mediated direct reversal repair, MutM and MutY catalyzed base excision repair, and DinB translesion synthesis on the BD-dA adducts in bacterial cells were investigated. BD-dA adducts showed the expected inhibition of DNA replication in vivo but were not substantively mutagenic in any of the genetic environments investigated. This result is in contrast with previous in vitro observations and opens the possibility that E. coli repair and bypass systems other than the ones studied here are able to minimize the mutagenic properties of BD-dA adducts.

Oncometabolites d- and l-2-Hydroxyglutarate Inhibit the AlkB Family DNA Repair Enzymes under Physiological Conditions.

Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.

Next-generation sequencing reveals the biological significance of the N(2),3-ethenoguanine lesion in vivo.

Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine.

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins.

Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments.