Substrate specificity of human thymine-DNA glycosylase on exocyclic cytosine adducts

The environmental carcinogen glycidaldehyde (GDA) and therapeutic chloroethylnitrosoureas (CNUs) can form hydroxymethyl etheno and ring-saturated ethano bases, respectively. The mutagenic potential of these adducts relies on their miscoding properties and repair efficiency. In this work, the ability of human thymine-DNA glycosylase (TDG) to excise 8-(hydroxymethyl)-3,N(4)-ethenocytosine (8-hm-varepsilonC) and 3,N(4)-ethanocytosine (EC) was investigated and compared with varepsilonC, a known substrate for TDG. When tested using defined oligonucleotides containing a single adduct, TDG is able to excise 8-hm-varepsilonC but not EC. The 8-hm-varepsilonC activity mainly depends on guanine pairing with the adduct. TDG removes 8-hm-varepsilonC less efficiently than varepsilonC but its activity can be significantly enhanced by human AP endonuclease 1 (APE1), a downstream enzyme in the base excision repair. TDG did not show any detectable activity toward EC when placed in various neighboring sequences, including the 5'-CpG site. Molecular modeling revealed a possible steric clash between the non-planar EC exocyclic ring and residue Asn 191 within the TDG active site, which could account for the lack of TDG activity toward EC. TDG was not active against the bulkier exocyclic adduct 3,N(4)-benzethenocytosine, nor the two adenine derivatives with same modifications as the cytosine derivatives, 7-hm-varepsilonA and EA. These findings expand the TDG substrate range and aid in understanding the structural requirements for TDG substrate specificity.     

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.     

AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo

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.     

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.     

Alkylpurine-DNA-N-glycosylase excision of 7-(hydroxymethyl)-1,N6-ethenoadenine, a glycidaldehyde-derived DNA adduct

Glycidaldehyde (GDA) is a bifunctional alkylating agent that has been shown to be mutagenic in vitro and carcinogenic in rodents. However, the molecular mechanism by which it exerts these effects is not established. GDA is capable of forming exocyclic hydroxymethyl-substituted etheno adducts on base residues in vitro. One of them, 7-(hydroxymethyl)-1,N6-ethenoadenine (7-hm-epsilonA), was identified as the principal adduct in mouse skin treated with GDA or a glycidyl ether. In this work, using defined oligonucleotides containing a site-specific 7-hm-epsilonA, the human and mouse alkylpurine-DNA-N-glycosylases (APNGs), responsible for the removal of the analogous 1,N6-ethenoadenine (epsilonA) adduct, are shown to recognize and excise 7-hm-epsilonA. Such an activity can be significantly modulated by both 5' neighboring and opposite sequence contexts. The efficiency of human or mouse APNG for excision of 7-hm-epsilonA is about half that, or similar to the excision of epsilonA, respectively. When human or mouse cell-free extracts were tested, however, the extent of 7-hm-epsilonA excision is dramatically lower than that for epsilonA, suggesting that, in the crude extracts, the APNG activities toward these two adducts are differentially affected. Using cell-free extracts from APNG deficient mice, this enzyme is shown to be the primary glycosylase excising 7-hm-epsilonA. A structural approach, using molecular modeling, was employed to examine how the structure of the 7-hm-epsilonA adduct affects DNA conformation, as compared to the epsilonA adduct. These novel substrate specificities could have both biological and structural implications.     

The role of the Escherichia coli mug protein in the removal of uracil and 3,N(4)-ethenocytosine from DNA.

The human thymine-DNA glycosylase has a sequence homolog in Escherichia coli that is described to excise uracils from U.G mismatches (Gallinari, P., and Jiricny, J. (1996) Nature 383, 735-738) and is named mismatched uracil glycosylase (Mug). It has also been described to remove 3,N(4)-ethenocytosine (epsilonC) from epsilonC.G mismatches (Saparbaev, M., and Laval, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8508-8513). We used a mug mutant to clarify the role of this protein in DNA repair and mutation avoidance. We find that inactivation of mug has no effect on C to T or 5-methylcytosine to T mutations in E. coli and that this contrasts with the effect of ung defect on C to T mutations and of vsr defect on 5-methylcytosine to T mutations. Even under conditions where it is overproduced in cells, Mug has little effect on the frequency of C to T mutations. Because uracil-DNA glycosylase (Ung) and Vsr are known to repair U.G and T.G mismatches, respectively, we conclude that Mug does not repair U.G or T.G mismatches in vivo. A defect in mug also has little effect on forward mutations, suggesting that Mug does not play a role in avoiding mutations due to endogenous damage to DNA in growing E. coli. Cell-free extracts from mug(+) ung cells show very little ability to remove uracil from DNA, but can excise epsilonC. The latter activity is missing in extracts from mug cells, suggesting that Mug may be the only enzyme in E. coli that can remove this mutagenic adduct. Thus, the principal role of Mug in E. coli may be to help repair damage to DNA caused by exogenous chemical agents such as chloroacetaldehyde.     

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.     

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.     

The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.     

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.     

Mismatch uracil glycosylase from Escherichia coli: a general mismatch or a specific DNA glycosylase?

The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U.G mismatches. Subsequently, 3,N4-ethenocytosine (epsilonC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme. The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme. Our results demonstrate that product release by the enzyme is very slow and hence comparing the "steady-state" parameters of the enzyme for different substrates is of limited use. Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions. Although the enzyme excised both epsilonC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis. Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate. This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism.     

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.     

Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.     

Cisplatin adducts inhibit 1,N(6)-ethenoadenine repair by interacting with the human 3-methyladenine DNA glycosylase

The human 3-methyladenine DNA glycosylase (AAG) is a repair enzyme that removes a number of damaged bases from DNA, including adducts formed by some chemotherapeutic agents. Cisplatin is one of the most widely used anticancer drugs. Its success in killing tumor cells results from its ability to form DNA adducts and the cellular processes triggered by the presence of those adducts in DNA. Variations in tumor response to cisplatin may result from altered expression of cellular proteins that recognize cisplatin adducts. The present study focuses on the interaction between the cisplatin intrastrand cross-links and human AAG. Using site-specifically modified oligonucleotides containing each of the cisplatin intrastrand cross-links, we found that AAG readily recognized cisplatin adducts. The apparent dissociation constants for the 1, 2-d(GpG), the 1,2-d(ApG), and the 1,3-d(GpTpG) oligonucleotides were 115 nM, 71 nM, and 144 nM, respectively. For comparison, the apparent dissociation constant for an oligonucleotide containing a single 1,N(6)-ethenoadenine (epsilonA), which is repaired efficiently by AAG, was 26 nM. Despite the affinity of AAG for cisplatin adducts, AAG was not able to release any of these adducts from DNA. Furthermore, it was demonstrated that the presence of cisplatin adducts in the reactions inhibited the excision of epsilonA by AAG. These data suggest a previously unexplored dimension to the toxicological response of cells to cisplatin. We suggest that cisplatin adducts could titrate AAG away from its natural substrates, resulting in higher mutagenesis and/or cell death because of the persistence of AAG substrates in DNA.     

Base J, found in nuclear DNA of Trypanosoma brucei, is not a target for DNA glycosylases

Base excision repair (BER) is an evolutionarily conserved system which removes altered bases from DNA. The initial step in BER is carried out by DNA glycosylases which recognize altered bases and cut the N-glycosylic bond between the base and the DNA backbone. In kinetoplastid flagellates, such as Trypanosoma brucei, the modified base beta-D-glucosyl-hydroxymethyluracil (J) replaces a small percentage of thymine residues, predominantly in repetitive telomeric sequences. Base J is synthesized at the DNA level via the precursor 5-hydroxymethyluracil (5-HmU). We have investigated whether J in DNA can be recognized by DNA glycosylases from non-kinetoplastid origin, and whether the presence of J and 5-HmU in DNA has required modifications of the trypanosome BER system. We tested the ability of 15 different DNA glycosylases from various origins to excise J or 5-HmU paired to A from duplex oligonucleotides. No excision of J was found, but 5-HmU was excised by AlkA and Mug from Escherichia coli and by human SMUG1 and TDG, confirming previous reports. In a combination of database searches and biochemical assays we identified several DNA glycosylases in T. brucei, but in trypanosome extracts we detected no excision activity towards 5-HmU or ethenocytosine, a product of oxidative DNA damage and a substrate for Mug, TDG and SMUG1. Our results indicate that trypanosomes have a BER system similar to that of other organisms, but might be unable to excise certain forms of oxidatively damaged bases. The presence of J in DNA does not require a specific modification of the BER system, as this base is not recognized by any known DNA glycosylase.     

Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli

Exocyclic alkylamino purine adducts, including N(2)-ethyldeoxyguanosine, N(2)-isopropyldeoxyguanosine, and N(6)-isopropyldeoxyadenosine, occur as a consequence of reactions of DNA with toxins such as the ethanol metabolite acetaldehyde, diisopropylnitrosamine, and diisopropyltriazene. However, there are few data addressing the biological consequences of these adducts when present in DNA. Therefore, we assessed the mutagenicities of these single, chemically synthesized exocyclic amino adducts when placed site-specifically in the supF gene in the reporter plasmid pLSX and replicated in Escherichia coli, comparing the mutagenic potential of these exocyclic amino adducts to that of O(6)-ethyldeoxyguanosine. Inclusion of deoxyuridines on the strand complementary to the adducts at 5' and 3' flanking positions resulted in mutant fractions of N(2)-ethyldeoxyguanosine and N(2)-isopropyldeoxyguanosine-containing plasmid of 1.4+/-0.5% and 5.7+/-2.5%, respectively, both of which were significantly greater than control plasmid containing deoxyuridines but no adduct (p=0.04 and 0.003, respectively). The mutagenicities of the three exocyclic alkylamino purine adducts tested were of smaller magnitude than O(6)-ethyldeoxyguanosine (mutant fraction=21.2+/-1.2%, p=0.00001) with the N(6)-isopropyldeoxyadenosine being the least mutagenic (mutant fraction=1.2+/-0.5%, p=0.13). The mutation spectrum generated by the N(2)-ethyl and -isopropyldeoxyguanosine adducts included adduct site-targeted G:C-->T:A transversions, adduct site single base deletions, and single base deletions three bases downstream from the adduct, which contrasted sharply with the mutation spectrum generated by the O(6)-ethyldeoxyguanosine lesion of 95% adduct site-targeted transitions. We conclude that N(2)-ethyl and -isopropyldeoxyguanosine are mutagenic adducts in E. coli whose mutation spectra differ markedly from that of O(6)-ethyldeoxyguanosine.     

Excision Repair of 2,5-Diaziridinyl-1,4-Benzoquinone (DZQ)-DNA Adduct by Bacterial and Mammalian 3-Methyladenine-DNA Glycosylases

The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.     

Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease

Adozelesin is a synthetic analog of the antitumor antibiotic CC-1065, which alkylates the N3 of adenine in the minor groove in a sequence-selective manner. Since the cytotoxic potency of a DNA alkylating agent can be modulated by DNA excision repair system, we investigated whether nucleotide excision repair (NER) and base excision repair (BER) enzymes are able to excise the bulky DNA adduct induced by adozelesin. The UvrABC nuclease and 3-methyladenine-DNA glycosylase, that exhibit a broad spectrum of substrate specificity, were selected as typical NER and BER enzymes, respectively. The adozelesin-DNA adduct was first formed in the radiolabeled restriction DNA fragment and its excision by purified repair enzymes was monitored on a DNA sequencing gel. The treatment of the DNA adduct with a purified UvrABC nuclease and sequencing gel analysis of cleaved DNA showed that UvrABC nuclease was able to incise the adozelesin adduct. The incision site corresponded to the general nuclease incision site. Excision of this adduct by 3-methyladenine-DNA glycosylases was determined following the treatment of the DNA adduct with a homogeneous recombinant bacterial, rat and human 3-methyladenine-DNA glycosylases. Abasic sites generated by DNA glycosyalses were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase (Fpg). Resolution of cleaved DNA on a sequencing gel showed that the DNA glycosylase from different sources could not release the N3-adenine adducts. A cytotoxicity assay using E. coli repair mutant strains showed that E. coli mutant strains defective in the uvrA gene were more sensitive to cell killing by adozelesin than E. coli mutant strain defective in the alkA gene or the wild type. These results suggest that the NER pathway seems to be the major excision repair system in protecting cells from the cytotoxicity of adozelesin.     

The Bacillus subtilis counterpart of the mammalian 3-methyladenine DNA glycosylase has hypoxanthine and 1,N6-ethenoadenine as preferred substrates

The AAG family of 3-methyladenine DNA glycosylases was initially thought to be limited to mammalian cells, but genome sequencing efforts have revealed the presence of homologous proteins in certain prokaryotic species as well. Here, we report the first molecular characterization of a functional prokaryotic AAG homologue, i.e. YxlJ, termed bAag, from Bacillus subtilis. The B. subtilis aag gene was expressed in Escherichia coli, and the protein was purified to homogeneity. As expected, B. subtilis Aag was found to be a DNA glycosylase, which releases 3-alkylated purines and hypoxanthine, as well as the cyclic etheno adduct 1,N(6)-ethenoadenine from DNA. However, kinetic analysis showed that bAag removed hypoxanthine much faster than human AAG with a 10-fold higher value for k(cat), whereas the rate of excision of 1, N(6)-ethenoadenine was found to be similar. In contrast, it was found that bAag removes 3-methyladenine and 3-methylguanine approximately 10-20 times more slowly than human AAG, and there was hardly any detectable excision of 7-methylguanine. It thus appears that bAag has a minor role in the repair of DNA alkylation damage and an important role in preventing the mutagenic effects of deaminated purines and cyclic etheno adducts in Bacillus subtilis.     

DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.