Chloroethylnitrosourea-derived ethano cytosine and adenine adducts are substrates for Escherichia coli glycosylases excising analogous etheno adducts

Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N4-ethanocytosine (EC) and 1,N6-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N4-ethenocytosine (epsilonC) and AlkA releases 1,N6-ethenoadenine (epsilonA). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a approximately 20-fold lower excision activity toward EC or EA than that toward their structurally analogous epsilonC or epsilonA adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the epsilonC base, which could account for the lower EC activity observed in this study.     

Hijacking of the human alkyl-N-purine-DNA glycosylase by 3,N4-ethenocytosine, a lipid peroxidation-induced DNA adduct

Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer.     

Interaction of tobacco mosaic virus protein with synthetic polynucleotides containing a fluorescent label: optical properties of poly(A,epsilonA) and poly(C,epsilonC) copolymers and energy migration from the tryptophan to 1,N6-ethenoadenine or 3,N4-ethenocytosine residues in RNP.

A spectrophotometric method for determination of the modification degrees and molar extinction coefficients for poly(A, epsilonA) and poly(C, epsilonC) copolymers has been developed. Dependence of some absorption and fluorescence parameters of the copolymers on the modification degree has been studied. Distribution of modified residues in copolymers differs from random and depends on modification conditions. Interaction between the TMV protein and copolymers has been investigated. The protein interacts with poly(A, epsilonA) of low or medium modification degree and displays no activity with respect to poly(epsilonA). On the contrary, introduction of epsilonC to the polynucleotide promotes complex formation between poly(C, epsilonC) and TMV protein. Analysis of the fluorescence emission and excitation spectra has revealed energy transfer from tryptophan to epsilonA or epsilon C in the RNP to occur and permits one to estimate the average distance between Trp (presumably Trp 52)o and the RNA base binding region in the virus to be 17 to 20 A.     

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.     

Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation

The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) α-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.     

Chemical rearrangement and repair pathways of 1,N6-ethenoadenine

1,N(6)-Ethenoadenine (epsilonA) is an exocyclic DNA adduct introduced to DNA by vinyl chloride and related compounds as well as in the consequence of oxidative stress and lipid peroxidation (LPO). This highly genotoxic DNA damage is chemically unstable and either depurinates or converts into pyrimidine ring-opened secondary lesions. We have studied the structures of derivatives formed during epsilonA chemical rearrangement and identified enzymes repairing one of the rearrangement products. Rearrangement involves a water molecule addition to the C(2)-N(3) bond of epsilonA, resulting in formation of pyrimidine ring-closed B1 product, which is in equilibrium with pyrimidine ring-opened B2 compound. B2 further deformylates to yield compound C. N-Glycosidic bond of compound C is unstable and C depurinates, yielding compound D. These secondary lesions are not repaired by alkylpurine DNA N-glycosylase, which excises the parental epsilon A. Compound B, when paired with thymine and cytosine is efficiently excised by Escherichia coli formamidopirymidine DNA N-glycosylase (Fpg), and thymine glycol DNA N-glycosylases from E. coli (Nth) and Saccharomyces cerevisiae (Ntg2). B is eliminated from B:G pair only by Nth and Ntg2 glycosylases, however none of the enzymes studied is excising B from B:A pair. This enables finishing of rearrangement, formation of AP sites and subsequently DNA strand breaks. During in vitro translesion synthesis, C is much easier bypassed by DNA polymerases, than compound B, and also than the parental epsilonA as well as than the AP site. This bypass beyond C proceeds mainly by misinsertion of adenine and guanine, or by insertion of thymine, the latter restoring the parental A:T pair. Alternatively, looping out of adducted nucleotide alone or with adjacent one generates one- or two-nucleotide deletions. This may explain the previously reported 20-fold higher mutagenic potency of product C in comparison to epsilon A in E. coli [Biochemistry 32 (1993) 12793].     

Sequence-specific p53 gene damage by chloroacetaldehyde and its repair kinetics in Escherichia coli

Oxidative stress and certain environmental carcinogens, e.g. vinyl chloride and its metabolite chloroacetaldehyde (CAA), introduce promutagenic exocyclic adducts into DNA, among them 1,N(6)-ethenoadenine (epsilonA), 3,N(4)-ethenocytosine (epsilonC) and N(2),3-ethenoguanine (epsilonG). We studied sequence-specific interaction of the vinyl-chloride metabolite CAA with human p53 gene exons 5-8, using DNA Polymerase Fingerprint Analysis (DPFA), and identified sites of the highest sensitivity. CAA-induced DNA damage was more extensive in p53 regions which revealed secondary structure perturbations, and were localized in regions of mutation hot-spots. These perturbations inhibited DNA synthesis on undamaged template. We also studied the repair kinetics of CAA-induced DNA lesions in E. coli at nucleotide resolution level. A plasmid bearing full length cDNA of human p53 gene was modified in vitro with 360 mM CAA and transformed into E. coli DH5alpha strain, in which the adaptive response system had been induced by MMS treatment before the cells were made competent. Following transformation, plasmids were re-isolated from transformed cultures 35, 40, 50 min and 1-24 h after transformation, and further subjected to LM-PCR, using ANPG, MUG and Fpg glycosylases to identify the sites of DNA damage. In adaptive response-induced E. coli cells the majority of DNA lesions recognized by ANPG glycosylase were removed from plasmid DNA within 35 min, while MUG glycosylase excised base modifications only within 50 min, both in a sequence-dependent manner. In non-adapted cells resolution of plasmid topological forms was perturbed, suggesting inhibition of one or more bacterial topoisomerases by unrepaired epsilon-adducts. We also observed delayed consequences of DNA modification with CAA, manifesting as secondary DNA breaks, which appeared 3 h after transformation of damaged DNA into E. coli, and were repaired after 24 h.     

Substrate specificity of human methylpurine DNA N-glycosylase

The activity of human methylpurine DNA N-glycosylase (hMPG) for major substrates was directly compared using two types of substrates, i.e., natural DNA and synthetic oligonucleotides. By the use of ARP assay detecting abasic sites in DNA, we first investigated the activity on the natural DNA substrates containing methylpurines, ethenopurines, or hypoxanthine (Hx) prepared by the conventional methods. After the treatment with hMPG, the amount of AP sites in methylated DNA was much higher than that in DNA containing ethenopurines or Hx. The oligodeoxynucleotide having a single 7-methylguanine (7-mG) was newly synthesized in addition to 1, N(6)-ethenoadenine (epsilonA)-, Hx-, and 8-oxoguanine-containing oligonucleotides. 7-mG was effectively excised by hMPG, though it might be less toxic than the other methylated bases with respect to mutagenesis and cell killing. The kinetic study demonstrated that k(cat)/K(m) ratios of the enzyme for epsilonA, Hx, and 7-mG were 2.5 x 10(-3), 1.4 x 10(-3), and 4 x 10(-4) min(-1) nM(-1), respectively. The oligonucleotides containing epsilonA effectively competed against 7-mG, while Hx substrates showed unexpectedly low competition. Concerning the effect of the base opposite damage, hMPG much preferred Hx.T to other Hx pairs, and epsilonA.C and epsilonA.A pairs were better substrates than epsilonA.T.     

Novel activity of Escherichia coli mismatch uracil-DNA glycosylase (Mug) excising 8-(hydroxymethyl)-3,N4-ethenocytosine, a potential product resulting from glycidaldehyde reaction

Glycidaldehyde is an industrial chemical which has been shown to be genotoxic in in vitro experiments and carcinogenic in rodent studies. It is a bifunctional alkylating agent capable of reacting with DNA to form exocyclic hydroxymethyl-substituted ethenobases. In this work, 8-(hydroxymethyl)-3,N4-etheno-2'-deoxycytidine (8-HM-epsilondC), a potential nucleoside derivative of glycidaldehyde, was synthesized using phosphoramidite chemistry and site-specifically incorporated into a defined 25-mer oligodeoxynucleotide. The 8-HM-epsilonC adduct is structurally related to 3,N4-ethenocytosine (epsilonC), a product of reaction with vinyl chloride or through lipid peroxidation. In Escherichia coli, epsilonC has been shown previously to be a primary substrate for the mismatch uracil-DNA glycosylase (Mug). In this study, we report that the same glycosylase also acts on 8-HM-epsilonC in an oligonucleotide duplex. The enzyme binds to the 8-HM-epsilonC-oligonucleotide to a similar extent as the epsilonC-oligonucleotide. The Mug excision activity toward 8-HM-epsilonC is approximately 2.5-fold lower than that toward the epsilonC substrate. Both activities can be stimulated up to approximately 2-fold higher by the addition of E. coli endonuclease IV. These two adducts, when mispaired with normal bases, were all excised from DNA by Mug with similar efficiencies. Structural studies using molecular simulations showed similar adjustment and hydrogen bonding pattern for both 8-HM-epsilonC*G and epsilonC*G pairs in oligomer duplexes. We believe that these findings may have biological and structural implications in defining the role of 8-HM-epsilonC in glycosylase recognition/repair.     

The type of DNA glycosylase determines the base excision repair pathway in mammalian cells.

The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.     

Base excision and DNA binding activities of human alkyladenine DNA glycosylase are sensitive to the base paired with a lesion

The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N(6)-ethenoadenine (epsilonA) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O(6)-methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and epsilonA paired with T showed that excision of Hx was about four times faster than epsilonA, whereas binding assays showed that the binding affinity was about five times greater for epsilonA than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for epsilonA. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.     

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

Evidence of complete cellular repair of 1,N6-ethenoadenine, a mutagenic and potential damage for human cancer, revealed by a novel method

1,N6-Ethenoadenine (epsilonA) is generated endogenously by lipid peroxidation and exogenously by tumorigenic industrial agents, vinyl chloride, and vinyl carbamate. epsilonA detected in human tissues causes mutation and is implicated in liver, colon and lung cancers. N-methyl purine DNA-glycosylase (MPG) is the only enzyme known so far to repair epsilonA. However, the mechanism of in vivo repair of epsilonA and the role of MPG remain enigmatic. Moreover, previous in vivo repair studies for DNA lesions, including epsilonA, focused only on the step of the removal of the base lesion without further insight into the completion of the repair process. This may be in part due to the unavailability of an appropriate in vivo quantitative method to evaluate complete BER process at the basal level. Our newly developed in vivo method is highly sensitive and involves phagemid M13mp18, containing epsilonA at a defined position. The complete repair events have been estimated by plaque assay in E. coli with the phagemids recovered from the human cells after cellular processing. We found that the detectable complete (removal and replacement of epsilonA with adenine) repair was observed only 18% in 16 h, but with the repair nearing completion within 24 h in colon cancer, HCT-116, cells. Moreover, MPG is the predominant enzyme for the BER process to remove epsilonA in mammalian cells. Although, the epsilonA is fairly a bulky adduct compared to other small BER substrate lesions, NER pathway is not involved in repair of this adduct. Furthermore, the epsilonA repair in vivo and in vitro is predominant in the G0/G1 phase of the cell cycle.     

Mismatch dependent uracil/thymine-DNA glycosylases excise exocyclic hydroxyethano and hydroxypropano cytosine adducts

Exocyclic adducts of DNA bases, such as etheno- and hydroxyalkano- ones, are generated by a variety of bifunctional agents, including endogenously formed products of lipid peroxidation. In this work we selectively modified cytosines in the 5'-d(TTT TTT CTT TTT CTT TTT CTT TTT T)-3' oligonucleotide using: chloroacetaldehyde to obtain 3,N(4)-alpha-hydroxyethano- (HEC) and 3,N(4)-etheno- (epsilonC), acrolein to obtain 3,N(4)-alpha-hydroxypropano- (HPC) and crotonaldehyde to obtain 3,N(4)-alpha-hydroxy-gamma-methylpropano- (mHPC) adducts of cytosine. The studied adducts are alkali-labile which results in oligonucleotide strain breaks at the sites of modification upon strong base treatment. The oligonucleotides carrying adducted cytosines were studied as substrates of Escherichia coli Mug, human TDG and fission yeast Thp1p glycosylases. All the adducts studied are excised by bacterial Mug although with various efficiency: epsilonC >HEC >HPC >mHPC. The yeast enzyme excises efficiently epsilonC>HEC>HPC, whereas the human enzyme excises only epsilonC. The pH-dependence curves of excision of eC, HEC and HPC by Mug are bell shaped and the most efficient excision of adducts occurs within the pH range of 8.6-9.6. The observed increase of excision of HEC and HPC above pH 7.2 can be explained by deprotonation of these adducts, which are high pK(a) compounds and exist in a protonated form at neutrality. On the other hand, since epsilonC is in a neutral form in the pH range studied, we postulate an involvement of an additional catalytic factor. We hypothesize that the enzyme structure undergoes a pH-induced rearrangement allowing the participation of Lys68 of Mug in catalysis via a hydrogen bond interaction of its epsilon-amino group with N(4) of the cytosine exocyclic adducts.     

Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

Background

In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question.

Methodology/Principal Findings

We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate.

Conclusions/Significance

A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a “searching” mode and “repair” mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.     

The pyrimidine ring-opened derivative of 1,N6-ethenoadenine is excised from DNA by the Escherichia coli Fpg and Nth proteins

It was previously shown that 1,N(6)-ethenoadenine (epsilonA) in DNA rearranges into a pyrimidine ring-opened derivative of 20-fold higher mutagenic potency in Escherichia coli (AB1157 lacDeltaU169) than the parental epsilonA (Basu, A. K., Wood, M. L., Niedernhofer, L. J., Ramos, L. A., and Essigmann, J. M. (1993) Biochemistry 32, 12793-12801). We have found that at pH 7.0, the stability of the N-glycosidic bond in epsilondA is 20-fold lower than in dA. In alkaline conditions, but also at neutrality, epsilondA depurinates or converts into products: epsilondA --> B --> C --> D. Compound B is a product of water molecule addition to the C(2)-N(3) bond, which is in equilibrium with a product of N(1)-C(2) bond rupture in epsilondA. Compound C is a deformylated derivative of ring-opened compound B, which further depurinates yielding compound D. Ethenoadenine degradation products are not recognized by human N-alkylpurine-DNA glycosylase, which repairs epsilonA. Product B is excised from oligodeoxynucleotides by E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Nth). Repair by the Fpg protein is as efficient as that of 7,8-dihydro-8-oxoguanine when the excised base is paired with dT and dC but is less favorable when paired with dG and dA. Ethenoadenine rearrangement products are formed in oligodeoxynucleotides also at neutral pH at the rate of about 2-3% per week at 37 degrees C, and therefore they may contribute to epsilonA mutations.     

8-(Hydroxymethyl)-3,N(4)-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.     

SOS and UVM pathways have lesion-specific additive and competing effects on mutation fixation at replication-blocking DNA lesions

Escherichia coli cells have multiple mutagenic pathways that are induced in response to environmental and physiological stimuli. Unlike the well-investigated classical SOS response, little is known about newly recognized pathways such as the UVM (UV modulation of mutagenesis) response. In this study, we compared the contributions of the SOS and UVM pathways on mutation fixation at two representative noninstructive DNA lesions: 3,N4-ethenocytosine (epsilonC) and abasic (AP) sites. Because both SOS and UVM responses are induced by DNA damage, and defined UVM-defective E. coli strains are not yet available, we first constructed strains in which expression of the SOS mutagenesis proteins UmuD' and UmuC (and also RecA in some cases) is uncoupled from DNA damage by being placed under the control of a heterologous lac-derived promoter. M13 single-stranded viral DNA bearing site-specific lesions was transfected into cells induced for the SOS or UVM pathway. Survival effects were determined from transfection efficiency, and mutation fixation at the lesion was analyzed by a quantitative multiplex sequence analysis procedure. Our results suggest that induction of the SOS pathway can independently elevate mutagenesis at both lesions, whereas the UVM pathway significantly elevates mutagenesis at epsilonC in an SOS-independent fashion and at AP sites in an SOS-dependent fashion. Although mutagenesis at epsilonC appears to be elevated by the induction of either the SOS or the UVM pathway, the mutational specificity profiles for epsilonC under SOS and UVM pathways are distinct. Interestingly, when both pathways are active, the UVM effect appears to predominate over the SOS effect on mutagenesis at epsilonC, but the total mutation frequency is significantly increased over that observed when each pathway is individually induced. These observations suggest that the UVM response affects mutagenesis not only at class 2 noninstructive lesions (epsilonC) but also at classical SOS-dependent (class 1) lesions such as AP sites. Our results add new layers of complexity to inducible mutagenic phenomena: DNA damage activates multiple pathways that have lesion-specific additive as well as suppressive effects on mutation fixation, and some of these pathways are not directly regulated by the SOS genetic network.     

AlkB demethylases flip out in different ways

Aberrant methylations in DNA are repaired by base excision repair (BER) and direct repair by a methyltransferase or by an oxidative demethylase of the AlkB type. Yang et al. [Nature 452 (2008) 961-966] have now solved the crystal structure of AlkB and human AlkB homolog 2 (hABH2) in complex with DNA using an ingenious crosslinking strategy to stabilize the DNA-protein complex. AlkB proteins have similar catalytic domains, but different DNA recognition motifs. Whereas AlkB mainly makes contact with the damaged strand, hABH2 makes numerous contacts with both strands. hABH2 flips out the damaged base and fills the vacant space by a hydrophobic amino acid residue similar to DNA glycosylases, essentially without distorting the double helix structure. In contrast, AlkB squeezes together the bases flanking the flipped-out base to maintain the base stack. This unprecedented flipping mechanism and the differences between AlkB and hABH2 in contacting the DNA strands explain their preferences for single stranded- and double stranded DNA, respectively.