[Show abstract][Hide abstract] ABSTRACT: Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.
Proceedings of the National Academy of Sciences 09/2015; 112(35):10890-5. DOI:10.1073/pnas.1502374112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 β 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.
Proceedings of the National Academy of Sciences 08/2015; 112(33). DOI:10.1073/pnas.1507709112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a viral entry receptor for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as ‘molecular sinks’ and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we created decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform.
Antiviral Research 01/2015; 116. DOI:10.1016/j.antiviral.2015.01.008 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.
Chemical Research in Toxicology 08/2014; 27(9). DOI:10.1021/tx5002817 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DinB, the E. coli translesion synthesis polymerase, has been shown to bypass several N2-alkylguanine adducts in vitro, including N2-furfurylguanine, the structural analog of the DNA adduct formed by the antibacterial agent nitrofurazone. Recently, it was demonstrated that the Fe(II)- and α-ketoglutarate-dependent dioxygenase AlkB, a DNA repair enzyme, can dealkylate in vitro a series of N2-alkyguanines, including N2-furfurylguanine. The present study explored, head to head, the in vivo relative contributions of these two DNA maintenance pathways (replicative bypass vs. repair) as they processed a series of structurally varied, biologically relevant N2-alkylguanine lesions: N2-furfurylguanine (FF), 2-tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine, and 2-ethylguanine. Each lesion was chemically synthesized and incorporated site-specifically into an M13 bacteriophage genome, which was then replicated in E. coli cells deficient or proficient for DinB and AlkB (4 strains in total). Biochemical tools were employed to analyze the relative replication efficiencies of the phage (a measure of the bypass efficiency of each lesion) and the base composition at the lesion site after replication (a measure of the mutagenesis profile of each lesion). The main findings were: 1) Among the lesions studied, the bulky FF and HF lesions proved to be strong replication blocks when introduced site-specifically on a single-stranded vector in DinB deficient cells. This toxic effect disappeared in the strains expressing physiological levels of DinB. 2) AlkB is known to repair N2-alkylguanine lesions in vitro; however, the presence of AlkB showed no relief from the replication blocks induced by FF and HF in vivo. 3) The mutagenic properties of the entire series of N2-alkyguanines adducts were investigated in vivo for the first time. None of the adducts were mutagenic under the conditions evaluated, regardless of the DinB or AlkB cellular status. Taken together, the data indicated that the cellular pathway to combat bulky N2-alkylguanine DNA adducts was DinB-dependent lesion bypass.
PLoS ONE 04/2014; 9(4):e94716. DOI:10.1371/journal.pone.0094716 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The AlkB enzyme is an Fe(II)- and α-ketoglutarate-dependent dioxygenase that repairs DNA alkyl lesions by a direct reversal of damage mechanism as part of the adaptive response in E. coli. The reported substrate scope of AlkB includes simple DNA alkyl adducts, such as 1-methyladenine, 3-methylcytosine, 3-ethylcytosine, 1-methylguanine, 3-methylthymine, and N6-methyladenine, as well as more complex DNA adducts, such as 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 1,N6-ethanoadenine. Previous studies have revealed, in a piecemeal way, that AlkB has an impressive repertoire of substrates. The present study makes two additions to this list, showing that alkyl adducts on the N2 position of guanine and N4 position of cytosine are also substrates for AlkB. Using high resolution ESI-TOF mass spectrometry, we show that AlkB has the biochemical capability to repair in vitro N2-methylguanine, N2-ethylguanine, N2-furan-2-yl-methylguanine, N2-tetrahydrofuran-2-yl-methylguanine, and N4-methylcytosine in ssDNA but not in dsDNA. When viewed together with previous work, the experimental data herein demonstrate that AlkB is able to repair all simple N-alkyl adducts occurring at the Watson–Crick base pairing interface of the four DNA bases, confirming AlkB as a versatile gatekeeper of genomic integrity under alkylation stress.
[Show abstract][Hide abstract] ABSTRACT: The AlkB enzyme is an Fe(II)- and α-ketoglutarate-dependent dioxygenase that repairs DNA alkyl lesions by a direct reversal of damage mechanism as part of the adaptive response in E. coli. The reported substrate scope of AlkB includes simple DNA alkyl adducts, such as 1-methyladenine, 3-methylcytosine, 3-ethylcytosine, 1-methylguanine, 3-methylthymine and N6-methyladenine, as well as more complex DNA adducts, such as 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 1,N6-ethanoadenine. Previous studies have revealed, in a piecemeal way, that AlkB has an impressive repertoire of substrates. The present study makes two additions to this list, showing that alkyl adducts on the N2 position of guanine and N4 position of cytosine are also substrates for AlkB. Using high resolution ESI-TOF mass spectrometry, we show that AlkB has the biochemical capability to repair in vitro N2-methylguanine, N2-ethylguanine, N2-furan-2-yl-methylguanine, N2-tetrahydrofuran-2-yl-methylguanine, and N4-methylcytosine. When viewed together with previous work, the experimental data herein demonstrate that AlkB is able to repair all simple N-alkyl adducts occurring at the Watson-Crick base pairing interface of the four DNA bases, confirming AlkB as a versatile gatekeeper of genomic integrity under alkylation stress.
Chemical Research in Toxicology 06/2013; 26(8). DOI:10.1021/tx400096m · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and α-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo.
Journal of the American Chemical Society 04/2012; 134(21):8896-901. DOI:10.1021/ja3010094 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3'-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.
[Show abstract][Hide abstract] ABSTRACT: DNA alkylation can cause mutations, epigenetic changes, and even cell death. All living organisms have evolved enzymatic and non-enzymatic strategies for repairing such alkylation damage. AlkB, one of the Escherichia coli adaptive response proteins, uses an α-ketoglutarate/Fe(II)-dependent mechanism that, by chemical oxidation, removes a variety of alkyl lesions from DNA, thus affording protection of the genome against alkylation. In an effort to understand the range of acceptable substrates for AlkB, the enzyme was incubated with chemically synthesized oligonucleotides containing alkyl lesions, and the reaction products were analyzed by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. Consistent with the literature, but studied comparatively here for the first time, it was found that 1-methyladenine, 1,N (6)-ethenoadenine, 3-methylcytosine, and 3-ethylcytosine were completely transformed by AlkB, while 1-methylguanine and 3-methylthymine were partially repaired. The repair intermediates (epoxide and possibly glycol) of 3,N (4)-ethenocytosine are reported for the first time. It is also demonstrated that O (6)-methylguanine and 5-methylcytosine are refractory to AlkB, lending support to the hypothesis that AlkB repairs only alkyl lesions attached to the nitrogen atoms of the nucleobase. ESI-TOF mass spectrometry is shown to be a sensitive and efficient tool for probing the comparative substrate specificities of DNA repair proteins in vitro.
Journal of nucleic acids 10/2010; 2010:369434. DOI:10.4061/2010/369434
[Show abstract][Hide abstract] ABSTRACT: The only Y-family DNA polymerase conserved among all domains of life, DinB and its mammalian ortholog pol kappa, catalyzes proficient bypass of damaged DNA in translesion synthesis (TLS). Y-family DNA polymerases, including DinB, have been implicated in diverse biological phenomena ranging from adaptive mutagenesis in bacteria to several human cancers. Complete TLS requires dNTP insertion opposite a replication blocking lesion and subsequent extension with several dNTP additions. Here we report remarkably proficient TLS extension by DinB from Escherichia coli. We also describe a TLS DNA polymerase variant generated by mutation of an evolutionarily conserved tyrosine (Y79). This mutant DinB protein is capable of catalyzing dNTP insertion opposite a replication-blocking lesion, but cannot complete TLS, stalling three nucleotides after an N(2)-dG adduct. Strikingly, expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB(+) background. We find that this phenomenon is not exclusively due to a futile cycle of abortive TLS followed by exonucleolytic reversal. Rather, gene products with roles in cell death and metal homeostasis modulate the toxicity of DinB(Y79L) expression. Together, these results indicate that DinB is specialized to perform remarkably proficient insertion and extension on damaged DNA, and also expose unexpected connections between TLS and cell fate.
Proceedings of the National Academy of Sciences 11/2009; 106(50):21137-42. DOI:10.1073/pnas.0907257106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Supersize me! Size-expanded DNA bases (xDNA) are able to encode natural DNA sequences in replication. In vitro experiments with a DNA polymerase show nucleotide incorporation opposite the xDNA bases with correct pairing. In vivo experiments using E. coli show that two xDNA bases (xA and xC, see picture) encode the correct replication partners.
[Show abstract][Hide abstract] ABSTRACT: The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N(6)-ethenoadenine (epsilonA). The crystal structures of AAG bound to epsilonA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In this study, we explore the substrate specificity of full-length and truncated Delta80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions but excised only a few of them. Single-turnover excision kinetics showed that in addition to the well-known epsilonA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with epsilonA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N(2)-ethenoguanine (1,N(2)-epsilonG), albeit weakly, from duplex DNA. Uracil was excised from both single- and double-stranded DNA, but only by full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on double-stranded DNA, AAG excised both epsilonA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.
[Show abstract][Hide abstract] ABSTRACT: The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo) is a major factor in the maintenance of the integrity of the human genome. There are estimates that this enzyme is responsible for eliminating as many as 10(5) potentially mutagenic and genotoxic lesions from the genome of each cell every day. Furthermore, inhibition of AP endonuclease may be effective in decreasing the dose requirements of chemotherapeutics used in the treatment of cancer as well as other diseases. Therefore, it is essential to accurately and directly characterize the enzymatic mechanism of AP endo. Here we describe specifically designed double-stranded DNA oligomers containing tetrahydrofuran (THF) with a 5'-phosphorothioate linkage as the abasic site substrate. Using H(2)(18)O during the cleavage reaction and leveraging the stereochemical preferences of AP endo and T4 DNA ligase for phosphorothioate substrates, we show that AP endo acts by a one-step associative phosphoryl transfer mechanism on a THF-containing substrate.
[Show abstract][Hide abstract] ABSTRACT: The genome and its nucleotide precursor pool are under sustained attack by radiation, reactive oxygen and nitrogen species, chemical carcinogens, hydrolytic reactions, and certain drugs. As a result, a large and heterogeneous population of damaged nucleotides forms in all cells. Some of the lesions are repaired, but for those that remain, there can be serious biological consequences. For example, lesions that form in DNA can lead to altered gene expression, mutation, and death. This perspective examines systems developed over the past 20 years to study the biological properties of single DNA lesions.
Chemical Research in Toxicology 02/2008; 21(1):232-52. DOI:10.1021/tx700292a · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O(6)-methylguanine (O(6)mG), which stably pairs with thymine during replication and thereby creates a promutagenic O(6)mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O(6)mG:T mismatches can lead to cell death or result in G:C-->A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O(6)mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O(6)mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O(6)mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O(6)mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O(6)mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O(6)mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.
DNA Repair 02/2008; 7(2):170-6. DOI:10.1016/j.dnarep.2007.09.003 · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA-damaging agents usually produce a vast collection of lesions within the genome. Analysis of these lesions from the structural and biological viewpoints is often complicated by the reality that some of the lesions are chemically fragile, leading to an even larger set of secondary and tertiary products. In an effort to deconvolute complex DNA-damage spectra, a strategy is presented whereby an oligonucleotide containing a specific target for chemical reaction is allowed to react with a DNA-damaging agent. A large collection of HPLC-resolvable modified oligonucleotides is generated, and chromatographically distinct members of the set are then individually characterized using chemical, spectroscopic, biochemical, and genetic probes. The biological component of this "chemical-biological fingerprinting" tool is the use of polymerase bypass in vivo in cells having defined replication status and quantitative and qualitative patterns of lesion-directed mutagenesis, as key properties that complement physical analysis of modified DNA. This approach was applied to the complex product spectrum generated by peroxynitrite in the presence of CO2; peroxynitrite is a powerful oxidizing and nitrating agent generated as part of immune response. An oligonucleotide containing the primary oxidation product, 7,8-dihydro-8-oxoguanine (8-oxoGua), which is highly susceptible to further oxidation and/or nitration, was treated with peroxynitrite. Using mass spectrometry, coelution with authentic standards, sensitivity to piperidine, recognition and strand cleavage by the DNA repair enzyme MutM, and mutagenicity and genotoxicity in vivo, a matrix was created that defined the properties of the secondary DNA lesions formed when 3-morpholinosydnonimine (SIN-1) delivered a low, constant flux of peroxynitrite to an oligonucleotide containing 8-oxoGua. Two lesions were identified as the diastereomers of spiroiminodihydantoin (Sp), which had been observed previously in nucleoside-based experiments employing SIN-1. A third lesion, triazine, was tentatively identified. However, in addition to these lesions, a number of secondary lesions were generated that had chemical-biological fingerprints inconsistent with that of any known 8-oxoGua-derived lesion described to date. In vitro experiments showed that while some of these newly characterized secondary lesions were removed from DNA by MutM, others were in fact very poor substrates for this repair enzyme. These 8-oxoGua-derived lesions also showed varying degrees of sensitivity to piperidine. Furthermore, all of the secondary lesions observed in this work were potently mutagenic and genotoxic in Escherichia coli. Therefore, while 8-oxoGua itself is nontoxic and only mildly mutagenic in repair-proficient cells, peroxynitrite reveals the promutagenic potential and triggers the covert nature of this DNA lesion.
Chemical Research in Toxicology 12/2007; 20(11):1718-29. DOI:10.1021/tx700273u · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno (ε)-lesions. AlkB, an iron-/α-ketoglutarate-dependent dioxygenase, repairs 1,N 6-ethenode-oxyadenosine (εA) and 3,N4- ethenodeoxycytidine (εC) in site-specifically modified single-stranded viral genomes in vivo and also protects the E. coli genome from the toxic effects of CAA. We examined the role of AlkB as a cellular defense against CAA by characterizing the frequencies, types, and distributions of mutations induced in the double-stranded supF gene of pSP189 damaged in vitro and replicated in AlkB-proficient (AlkB+) and AlkB-deficient (AlkB-) E. coli. AlkB reduced mutagenic potency and increased the survival of CAA-damaged plasmids. Toxicity and mutagenesis data were benchmarked to levels of ε-adducts and DNA strand breaks measured by LC-MS/MS and a plasmid nicking assay. CAA treatment caused dose-dependent increases in εA, εC, and 1,N2-ethenodeoxyguanosine (1,N2-εG) and small increases in strand breaks and abasic sites. Mutation frequency increased in plasmids replicated in both AlkB+ and AlkB- cells; however, at the maximum CAA dose, the mutation frequency was 5-fold lower in AlkB+ than in AlkB- cells, indicating that AlkB protected the genome from CAA lesions. Most induced mutations in AlkB- cells were G:C to A:T transitions, with lesser numbers of G:C to T:A transversions and A:T to G:C transitions. G:C to A:T and A:T to G:C transitions were lower in AlkB+ cells than in AlkB- cells. Mutational hotspots at G122, G123, and G160 were common to both cell types. Three additional hotspots were found in AlkB- cells (C133, T134, and G159), with a decrease in mutation frequency and change in mutational signature in AlkB+ cells. These results suggest that the AlkB protein contributes to the elimination of exocyclic DNA base adducts, suppressing the toxic and mutagenic consequences induced by this damage and contributing to genetic stability.
Chemical Research in Toxicology 09/2007; 20(8):1075-83. DOI:10.1021/tx700167v · 3.53 Impact Factor