Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Nickel impairs the repair of UV- and MNNG-damaged DNA

Nickel(II) is reported to be genotoxic, but the mechanisms underlying its genotoxicity are largely unknown. It can interfere with DNA repair and this may contribute to its genotoxicity. We studied the effect of nickel chloride on the repair of DNA damaged by UV radiation or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in human lymphocytes using the alkaline comet assay. Nickel(II) at 1 microM caused an accumulation of DNA breaks during repair incubation, which could follow from the inhibition of the polymerization/ligation step of UV-damaged DNA repair. On the other hand, nickel(II) inhibited the formation of transient DNA breaks brought by the repair process after incubation with MNNG at 5 microM, which might follow from interference with the recognition/incision step of excision repair. Additionally, nickel at 1 microM inhibited the activity of formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (Alk A), enzymes involved in DNA excision repair. A decrease in endonuclease III (Endo III) activity was observed at 2 and 5 microM of nickel chloride. Our results suggest that nickel(II) at non-cytotoxic concentrations can inhibit various steps of DNA excision repair, and this may contribute to its genotoxicity.     

In vitro host cell reactivation of alkylated bacteriophage T7 deoxyribonucleic acid by repair-deficient strains of Escherichia coli.

An in vitro system capable of packaging bacteriophage T7 deoxyribonucleic acid (DNA) into phage heads to form viable phage particles has been used to monitor the biological consequences of DNA dam aged by alkylating agents, and an in vitro DNA replication system has been used to examine the ability of alkylated T7 DNA to serve as template for DNA synthesis. The survival of phage resulting from in vitro packaging of DNA preexposed to various concentrations of methyl methane sulfonate or ethyl methane sulfonate closely paralleled the in vivo situation, in which intact phage were exposed to the alkylating agents. Host factors responsible for survival of alkylated T7 have been examined by using wild-type strains of EScherichia coli and mutants deficient in DNA polymerase I (polA) or 3-methyladenine-DNA glycosylase (tag). For both in vivo and in vitro situations, a deficiency in 3-methyladenine-DNA glycosylase dramatically reduced phage survival relative to that in the wild type, whereas a deficiency in DNA polymerase I had an intermediate effect. Furthermore, when the tag mutant was used as an indicator strain, phage survival was enhanced when alkylated DNA was packaged with extracts prepared from a wild-type strain in place of the tag mutant or by complementing a tag extract with an uninfected tag+ extract, indicating in vitro repair during packaging.     

Escherichia coli, Saccharomyces cerevisiae, rat and human 3-methyladenine DNA glycosylases repair 1,N6-ethenoadenine when present in DNA.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which generate various ethenobases in DNA. It has been reported that 1,N6-ethenoadenine (epsilon A) is excised by a DNA glycosylase present in human cell extracts, whereas protein extracts from Escherichia coli and yeast were devoid of such an activity. We confirm that the human 3-methyladenine-DNA glycosylase (ANPG protein) excises epsilon A residues. This finding was extended to the rat (ADPG protein). We show, at variance with the previous report, that pure E.coli 3-methyladenine-DNA glycosylase II (AlkA protein) as well as its yeast counterpart, the MAG protein, excise epsilon A from double stranded oligodeoxynucleotides that contain a single epsilon A. Both enzymes act as DNA glycosylases. The full length and the truncated human (ANPG 70 and 40 proteins, respectively) and the rat (ADPG protein) 3-methyladenine-DNA glycosylases activities towards epsilon A are 2-3 orders of magnitude more efficient than the E.coli or yeast enzyme for the removal of epsilon A. The Km of the various proteins were measured. They are 24, 200 and 800 nM for the ANPG, MAG and AlkA proteins respectively. These three proteins efficiently cleave duplex oligonucleotides containing epsilon A positioned opposite T, G, C or epsilon A. However the MAG protein excises A opposite cytosine much faster than opposite thymine, guanine or adenine.     

Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase

Two Escherichia coli K12 mutants defective in 3-methyladenine-DNA glycosylase have been isolated following mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine. The mutants, which are of independent origin and have been designated tag-1 and tag-2, contain greatly reduced amounts of 3-methyladenine-DNA glycosylase activity in cell-free extracts. The defect in the tag-1 strain is observed at 43 °C but not at 30 °C, and a partially purified enzyme from this strain is unusually heat-labile, indicating that the defect in the tag-1 strain is due to a mutation in the structural gene for 3-methyladenine-DNA glycosylase.We have shown that 3-methyladenine-DNA glycosylase is responsible for the rapid removal of 3-methyladenine from the DNA of E. coli cells treated with monofunctional alkylating agents. The active release of this base is greatly impaired in the mutant strains. Both tag mutant strains are abnormally sensitive to killing by monofunctional alkylating agents and are defective in the host cell reactivation of methyl methanesulphonate-treated bacteriophage A. The tag mutation does not confer an increased sensitivity to ultraviolet or X-irradiation, and host cell reactivation of irradiated λ is normal in these strains. Further, there was no increase in the rate of spontaneous mutation in a tag strain.Three-factor transductional crosses with nalA and nrdA have shown that the tag-2 mutation is located at 47.2 minutes on the map of the E. coli K12 chromosome. In the mapping experiments, the tag-1 mutation behaved differently and appeared to be located at 43 to 46 minutes, in a closely situated but non-adjacent gene. Possible implications of the non-identity of the tag-1 and tag-2 mutations are discussed.     

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.     

Cloning of Micrococcus luteus 3-methyladenine-DNA glycosylase genes in Escherichia coli

The 3-methyladenine-DNA glycosylase (m3ADG) excises 3-methyladenine (m3A) residues formed in DNA after treatment with alkylating agents. In Escherichia coli, the repair of this type of damage depends on the products of the genes tagA and/or alkA, which code for m3ADG I (20 kDa) and II (30 kDa), respectively. The tagA- and alkA--single mutants are sensitive to alkylating agents, the double mutant much more so. We have cloned two genes of Micrococcus luteus that can partly substitute the function of the E. coli tagA- and alkA- genes. An M. luteus genome bank was made by shotgun cloning of EcoRI + BamHI-digested DNA into pBR322. Two hybrid plasmids were identified that confer methylmethane sulfonate (MMS) resistance to the tagA- ada+ mutant and a capacity to reactivate MMS-treated bacteriophage lambda. Each hybrid plasmid directed the synthesis of 21-kDa m3ADG in E. coli tagA- ada-, which were not inhibited by 4 mM m3A. However, the restriction maps of the two cloned genes were different, and they showed no sequence homology as judged by the lack of cross hybridization.     

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.     

Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and its biologically active beta-oxidation product phenylacetic acid mustard (PAM; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromatic alkylators. CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant. The chemical structures indicate that CLB and PAM are mutagenic, teratogenic and carcinogenic, but the mode of action has remained obscure. We have investigated the biological effects of CLB and PAM with DNA repair-deficient Escherichia coli strains. In contrast to MNNG (N-methyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E. coli, but permeabilization of the outer membrane of the cells through use of polymyxin B nonapeptide (PMBN) rendered them susceptible to these compounds. The importance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacking O(6)-methylguanine-DNA methyltransferase I and II (ada ogt). Similarly, the protective role of base excision repair (BER) was substantiated by demonstration of an even more increased susceptibility to CLB and PAM of cells lacking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1). Cells deficient in mismatch repair (mutS) appeared to be slightly more sensitive than normal cells to CLB and PAM, although no such sensitivity to MNNG was observed. This implicates the role of mismatches in CLB- and PAM-related cytotoxicity. It is generally believed that bifunctional alkylating agents, like CLB and PAM, exert their cytotoxic action via DNA cross-linking. Our results with O(6)-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient cells indicate that removal of the adducts prior to the formation of cross-links is an important mechanism maintaining cell viability. We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E. coli cells, whose outer membrane is not naturally permeable to mutagens or other interesting compounds.     

Regulation of expression of the cloned ada gene in Escherichia coli

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned in multicopy plasmids. O6-Methylguanine-DNA methyltransferase and 3-methyladenine-DNA glycosylase II, which are known to be inducible as part of the adaptive response, were produced in ada- cells bearing ada+ plasmids, even without treatment with alkylating agents. When such cells had been treated with methyl methanesulfonate, even higher levels of the enzyme activities were produced. Maxicell experiments revealed that the ada gene codes for a polypeptide with a molecular weight of 38 000. We constructed a hybrid plasmid carrying an ada'-lacZ' fused gene, with the proper control region for ada expression. beta-Galactosidase synthesis from the fused gene was strongly induced only when cells were treated with low doses of methylating agents, but was weakly induced with relatively high doses of ethylating agents. The induction was autogenously regulated by the ada gene product, in a positive manner.     

Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases. An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions. These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination.     

A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.     

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 formami-dopyrimidine-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.     

Purification and characterization of human 3-methyladenine-DNA glycosylase.

A human cDNA coding sequence for a 3-methyladenine-DNA glycosylase was expressed in Escherichia coli. In addition to the full-length 3-methyladenine-DNA glycosylase coding sequence, two other sequences (resulting from differential RNA splicing and the truncated anpg cDNA) derived from that sequence were also expressed. All three proteins were purified to physical homogeneity and their N-terminal amino acid sequences are identical to those predicted by the nucleic acid sequences. The full-length protein has 293 amino acids coding for a protein with a molecular mass of 32 kDa. Polyclonal antibodies against one of the proteins react with the other two proteins, and a murine 3-methyladenine-DNA glycosylase, but not with several other E. coli DNA repair proteins. All three proteins excise 3-methyl-adenine, 7-methylguanine, and 3-methylguanine as well as ethylated bases from DNA. The activities of the proteins with respect to ionic strength (optimum 100 mM KCl), pH (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: Km 9 nM and kcat 10 min-1, 7-methylguanine: Km 29 nM and kcat 0.38 min-1) are comparable. In contrast to these results, however, the thermal stability of the full-length and splicing variant proteins at 50 degrees C is less than that of the truncated protein.     

Free radical scavengers can modulate the DNA-damaging action of alloxan

Alloxan can generate diabetes in experimental animals and its action can be associated with the production of free radicals. It is therefore important to check how different substances often referred to as free radical scavengers may interact with alloxan, especially that some of these substance may show both pro- and antioxidant activities. Using the alkaline comet assay we showed that alloxan at concentrations 0.01-50 microM induced DNA damage in normal human lymphocytes in a dose-dependent manner. Treated cells were able to recover within a 120-min incubation. Vitamins C and E at 10 and 50 microM diminished the extent of DNA damage induced by 50 microM alloxan. Pre-treatment of the lymphocytes with a nitrone spin trap, alpha-(4-pyridil-1-oxide)- N-t-butylnitrone (POBN) or ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which mimics glutathione peroxides, reduced the alloxan-evoked DNA damage. The cells exposed to alloxan and treated with formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results confirmed that free radicals are involved in the formation of DNA lesions induced by alloxan. The results also suggest that alloxan can generate oxidized DNA bases with a preference for purines and contribute to their alkylation.     

Human 3-methyladenine-DNA glycosylase: Demonstration of a stimulatory factor

Cultured human lymphoblasts contain a component that stimulates 3-methyladenine-DNA glycosylase, resulting in increased removal of 3-methyladenine from alkylated DNA. Increased release of other methylated bases was not detected by high-pressure liquid chromatography. Separation of the component and 3-methyladenine-DNA glycosylase during enzyme purification results in a loss of glycosylase activity. Stimulation of glycosylase activity can be demonstrated by recombination of the separated component with a partially purified lymphoblast enzyme fraction.     

3-methyladenine-DNA glycosylase II: the crystal structure of an AlkA-hypoxanthine complex suggests the possibility of product inhibition

Escherichia coli (E. coli) protein 3-methyladenine-DNA glycosylase II (AlkA) functions primarily by removing alkylation damage from duplex and single stranded DNA. A crystal structure of AlkA was refined to 2.0 A resolution. This structure in turn was used to refine an AlkA-hypoxanthine (substrate) complex structure to 2.4 A resolution. The complex structure shows hypoxanthine located in AlkA's active site stacked between residues W218 and Y239. The structural analysis of the AlkA and AlkA-hypoxanthine structures indicate that free hypoxanthine binding in the active site may inhibit glycosylase activity.     

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.