[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; · 3.67 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. · 10.68 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.
[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. · 9.74 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.
Angewandte Chemie International Edition 06/2009; 48(25):4524-7. · 13.73 Impact Factor
[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: 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. · 4.27 Impact Factor
[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. · 3.67 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. · 3.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were <or=1.9% in wild-type E. coli. In contrast to OxodG bypass, Fapy.dG mutation frequencies were unaffected by carrying out experiments in mutM/mutY cells. Overall, these experiments suggest that Fapy.dG is at most weakly mutagenic in E. coli. Steady-state kinetic experiments using the Klenow fragment of DNA polymerase I from E. coli suggest that a low dA misincorporation frequency opposite Fapy.dG and inefficient extension of a Fapy.dG:dA base pair work synergistically to minimize the levels of G --> T transversions.
[show abstract][hide abstract] ABSTRACT: 2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno ()-lesions. AlkB, an iron-/alpha-ketoglutarate-dependent dioxygenase, repairs 1, N (6)-ethenodeoxyadenosine (A) and 3, N (4)-ethenodeoxycytidine (C) 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 -adducts and DNA strand breaks measured by LC-MS/MS and a plasmid nicking assay. CAA treatment caused dose-dependent increases in A, C, and 1, N (2)-ethenodeoxyguanosine (1, N (2)-G) 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. · 3.67 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.
Journal of Biological Chemistry 05/2007; 282(17):12741-8. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The DNA damage product 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG) is a commonly used biomarker of oxidative stress. The mutagenic potential of this DNA lesion is mitigated in Escherichia coli by multiple enzymes. One of these enzymes, MutY, excises an A mispaired with 8-oxoG as part of the process to restore the original G:C base pair. However, numerous studies have shown that 8-oxoG is chemically labile toward further oxidation. Here, we examine the activity of MutY on the 8-oxoG oxidation products guanidinohydantoin (Gh), two diastereomers of spiroiminodihydantoin (Sp1 and Sp2), oxaluric acid (Oa), and urea (Ur). Single-stranded viral genomes containing a site-specific lesion were constructed and replicated in E. coli that are either proficient in DNA repair or that lack MutY. These lesions were found previously to be potently mutagenic in repair competent bacteria, and we report here that these 8-oxoG-derived lesions are equally miscoding when replicated in E. coli lacking MutY; no significant change in mutation identity or frequency is observed. Interestingly, however, in the presence of MutY, Sp1 and Sp2 are more toxic than in cells lacking this repair enzyme.
[show abstract][hide abstract] ABSTRACT: 1,N(6)-ethanoadenine (EA) forms through the reaction of adenine in DNA with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic used to combat various brain, head, and neck tumors. Previous studies of the toxic and mutagenic properties of the DNA adduct EA have been limited to in vitro experiments using mammalian polymerases and have revealed the lesion to be both miscoding and genotoxic. This work explores lesion bypass and mutagenicity of EA replicated in vivo and demonstrates that EA is neither toxic nor mutagenic in wild-type Escherichia coli. Although the base excision repair glycosylase enzymes of both humans and E. coli possess a weak ability to act on the lesion in vitro, an in vivo repair pathway has not yet been demonstrated. Here we show that an enzyme mechanistically unrelated to DNA glycosylases, the adaptive response protein AlkB, is capable of acting on EA via its canonical mechanism of oxidative dealkylation. The reaction alleviates the unrepaired adduct's potent toxicity through metabolism at the C8 position (attached to N1 of adenine), producing a nontoxic and weakly mutagenic N(6) adduct. AlkB is shown here to be a geno-protective agent that reduces the toxicity of DNA damage by converting the primary adduct to a less toxic secondary product.
Proceedings of the National Academy of Sciences 02/2007; 104(3):755-60. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA damage, if left unrepaired, may hinder translesion synthesis, leading to cytotoxicity, and instruct a DNA polymerase to incorporate an incorrect incipient base opposite the damage, leading to mutagenicity. This chapter describes technology used to measure quantitatively the degree to which a specific type of DNA damage impedes DNA replication. The technology also quantifies the mutation frequency and specificity of such damage after replication within cells. If cells with defined defects in DNA repair are used as hosts for replication, one can pinpoint the specific enzymes or pathways of repair that are operative on specific types of DNA damage.
Methods in Enzymology 02/2006; 408:1-15. · 2.00 Impact Factor
[show abstract][hide abstract] ABSTRACT: Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase eta (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD'2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase kappa, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10-15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the 'steric gate' residue of DinB (Phe13 --> Val) and that of its archaeal homologue Dbh (Phe12 --> Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.
[show abstract][hide abstract] ABSTRACT: We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.
Proceedings of the National Academy of Sciences 12/2005; 102(44):15803-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oxidative stress converts lipids into DNA-damaging agents. The genomic lesions formed include 1,N(6)-ethenoadenine (epsilonA) and 3,N(4)-ethenocytosine (epsilonC), in which two carbons of the lipid alkyl chain form an exocyclic adduct with a DNA base. Here we show that the newly characterized enzyme AlkB repairs epsilonA and epsilonC. The potent toxicity and mutagenicity of epsilonA in Escherichia coli lacking AlkB was reversed in AlkB(+) cells; AlkB also mitigated the effects of epsilonC. In vitro, AlkB cleaved the lipid-derived alkyl chain from DNA, causing epsilonA and epsilonC to revert to adenine and cytosine, respectively. Biochemically, epsilonA is epoxidized at the etheno bond. The epoxide is putatively hydrolyzed to a glycol, and the glycol moiety is released as glyoxal. These reactions show a previously unrecognized chemical versatility of AlkB. In mammals, the corresponding AlkB homologs may defend against aging, cancer and oxidative stress.