Activities involved in base excision repair of bacteriophage T4 and lambda DNA in vivo

Summary The in vivo excision repair functions of Escherichia coli exonuclease III and 3-methyladenine DNA glycosylase I, and bacteriophage T4 pyrimidine dimer-DNA glycosylase were investigated. Following exposure of bacteriophage T4 or lambda to methyl methanesulfonate or ultraviolet irradiation, survival was determined by plating on E. coli have various genetic backgrounds. Although exonuclease III was shown to participate in base excision repair initiated by 3-methyladenine DNA glcosylase I, it had no detectable role in base excision repair initiated by the T4 pyrimidine dimer-DNA glycosylase. Despite its 3 apurinic/apyrimidinic endonuclease activity in vitro, T4 pyrimidine dimer-DNA glycosylase, even in large quantities, did not complement mutants defective in exonuclease III in the repair of apurinic sites generated by 3-methyladenine DNA glycosylase I in vivo.     

Purification and characterization of 3-methyladenine DNA glycosylase I from Escherichia coli.

We have purified 3-methyladenine DNA glycosylase I from Escherichia coli to apparent physical homogeneity. The enzyme preparation produced a single band of Mr 22,500 upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis in good agreement with the molecular weight deduced from the nucleotide sequence of the tag gene (Steinum, A.-L. and Seeberg, E. (1986) Nucl. Acids Res. 14, 3763-3772). HPLC confirmed that the only detectable alkylation product released from (3H)dimethyl sulphate treated DNA was 3-methyladenine. The DNA glycosylase activity showed a broad pH optimum between 6 and 8.5, and no activity below pH 5 and above pH 10. MgSO4, CaCl2 and MnCl2 stimulated enzyme activity, whereas ZnSO4 and FeCl3 inhibited the enzyme at 2 mM concentration. The enzyme was stimulated by caffeine, adenine and 3-methylguanine, and inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 3-methyladenine. The enzyme showed no detectable endonuclease activity on native, depurinated or alkylated plasmid DNA. However, apurinic sites were introduced in alkylated DNA as judged from the strand breaks formed by mixtures of the tag enzyme and the bacteriophage T4 denV enzyme which has apurinic/apyrimidinic endonuclease activity. It was calculated that wild-type E. coli contains approximately 200 molecules per cell of 3-methyladenine DNA glycosylase I.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.     

Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204–213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5–10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.     

Apurinic endonuclease from Saccharomyces cerevisiae.

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.     

Partial purification and characterization of the major AP endonuclease from Chlamydomonas reinhardi

The major AP endonuclease from Chlamydomonas reinhardi has been partially purified and characterized. The enzyme has a molecular weight of about 38 000 as measured by molecular sieving. There is an absolute requirement for a divalent cation, with magnesium being better than manganese. The activity is stimulated by dithiothreitol and Triton X-100. The activity is sensitive to ionic strength, as 50 mM NaCl or KCl results in 70% inhibition. The enzyme is specific for apurinic and apyrimidinic (AP) sites and does not cleave DNA that has been damaged by ultraviolet light, methyl methanesulfonate, osmium tetroxide or sodium bisulfite. There is no deficiency in the AP endonuclease activity in extracts prepared from two mutants of Chlamydomonas that are sensitive to both ultraviolet light and methyl methanesulfonate. There was no evidence for induction of AP endonuclease after exposure of the cells to methyl methanesulfonate.     

Evidence for unique DNA repair activity encoded by a cloned Serratia marcescens gene: suppression of Escherichia coli mutations that reduce repair of alkylated DNA.

A recombinant plasmid containing a Serratia marcescens DNA repair gene has been analyzed biochemically and genetically in Escherichia coli mutants deficient for repair of alkylated DNA. The cloned gene suppressed sensitivity to methyl methanesulfonate of an E. coli strain deficient in 3-methyladenine DNA glycosylases I and II (i.e., E. coli tag alkA) and two different E. coli recA mutants. Attempts to suppress the methyl methanesulfonate sensitivity of the E. coli recA mutant by using the cloned E. coli tag and alkA genes were not successful. Southern blot analysis did not reveal any homology between the S. marcescens gene and various known E. coli DNA repair genes. Biochemical analysis with the S. marcescens gene showed that the encoded DNA repair protein liberated 3-methyladenine from alkylated DNA, indicating that the DNA repair molecular is an S. marcescens 3-methyladenine DNA glycosylase. The ability to suppress both types of E. coli DNA repair mutations, however, suggests that the S. marcescens gene is a unique bacterial DNA repair gene.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Repair of 3-methyladenine and 7-methylguanine in nuclear DNA of Chlamydomonas: Requirement for protein synthesis

The removal of 3-methyladenine and 7-methylguanine from nuclear DNA was determined following exposure of Chlamydomonas reinhardi to methyl methanesulfonate (MMS). The amount of 3-methyladenine in DNA was determined using an extract from Micrococcus luteus that has a 3-methyladenine-DNA glycosylase. The amount of 7-methylguanine was estimated by heating the DNA for 30 min at 70° followed by alkaline hydrolysis of the resulting apurinic sites. The molecular weight of the DNA was determined using alkaline sucrose gradients. The 3-methyladenine is removed with a half-life of 2–3 h whereas the 7-methylaguanine is removed with a half-life of 10–12 h. The rate of removal of the 7-methylguanine is more than an order of magnitude faster than the estimated non-enzymatic hydrolysis rate indicating the probability of enzymatic repair. Addition of cycloheximide immediately after MMS treatment inhibits the removal of 3-methyladenine and 7-methylguanine from DNA. If cycloheximide is added 1.5 h after treatment with MMS, there is much less inhibition of the removal of 3-methyladenine. These results are interpreted to mean that MMS induces the synthesis of 1 or more proteins that are required for the repair of 3-methyladenine from Chlamydomonas DNA.     

A new Schizosaccharomyces pombe base excision repair mutant,nth1, reveals overlapping pathways for repair of DNA base damage

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.     

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.     

3-Methyladenine-DNA glycosylase: a probe for determining alkylation damage and repair in human fibroblasts

3-Methyladenine-DNA glycosylase was partially purified from human lymphoblasts and used as an enzymatic probe to assay the amounts of 3-methyladenine in DNA from cultured human fibroblasts after treatment with dimethyl sulfate. Aside from this specific alkylation product, the total number of alkylated bases was estimated after depurination by heating. Both enzyme-induced and heat-induced apurinic sites were converted to strand breaks and estimated after alkaline sucrose-gradient sedimentation. The results indicate that 3-methyladenine in cultured human fibroblasts is rapidly excised, with a half-life of about 2 hours. The rest of the alkylated purines (mostly 7-methylguanine) are removed much more slowly, with a half-life of about 20 hours.     

Human cancer, carcinogenic exposures and mutation spectra

Exposure of mammalian cells to alkylating agents causes transfer of alkyl groups to N- as well as O-atoms of DNA bases. Especially the O-alkylated G and T bases have strong mutagenic properties, since they are capable of mispairing during replication. The mutagenic potential of N-alkylbases is less clear although specific base excision repair (BER) pathways exist which remove those lesions from the DNA. We investigated the relative contribution of N-alkylations to mutation induction at the Hprt gene in cultured Chinese hamster ovary cells (CHO). To this end BER activity in CHO cells was modulated by introduction of an expression vector carrying the rat N-alkylpurine-DNA glycosylase (APDG) gene, which codes for a glycosylase that is able to remove 3-methyladenine and 7-methylguanine from DNA thereby generating apurinic sites. Upon selection of a CHO clone which 10 times overproduced APDG compared to control CHO cells, mutation induction, the mutational spectrum, and cell survival were determined in both cell lines following treatment with methyl methanesulfonate (MMS). The results show that over-expression of APDG renders CHO cells more sensitive for mutation induction as well as cytotoxicity induced by MMS. The involvement of apurinic sites in induction of base pair changes at positions where 3-methyladenine was induced is inferred from the observation that the mutational spectrum of MMS-induced mutations in APDG-CHO cells showed twice as much base pair changes at AT base pairs (33.3%) compared to the spectrum of MMS-induced mutations in CHO-control cells (15.8%).     

Apurinic acid endonuclease activity from mouse epidermal cells.

An endonuclease activity making single-strand breaks into depurinated and alkylated DNA has been purified 500-fold from carcinogen-transformed mouse epidermal cells. The enzyme was active only at apurinic/apyrimidinic sites, regardless of whether they were produced by heating at an acidic pH or by alkylation with the ultimate carcinogen MeSO2OMe. The enzyme did not act on native DNA nor on ultraviolet-induced pyrimidine-dimers nor on steric distortions caused by modification of DNA with the carcinogen (Ac)2ONFln. The enzyme was active in the presence of 1 mM EDTA; however, at pH 7.4 optimal conditions were: 6mM MgCl2 and 40--120 mM KCl or 10--40 mM potassium phosphate. The enzyme eluted from hydroxyapatite, phosphocellulose and heparin-cellulose between 100--250 mM potassium phosphate but did not bind to DEAE-cellulose. Using four chromatographic steps the endonuclease was obtained free of exonuclease, demethylase and DNA glycosylase activity specific for DNA bases methylated with MeSO2OMe or MeNOUr. The molecular weight was 31 000 +/- 3000 as calculated from the diffusion coefficient (8.2 x 10-7 cm2/s) and the sedimentation value (2.7 S).     

Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated DNA.     

Structure and expression of the alkA gene of Escherichia coli involved in adaptive response to alkylating agents

Nucleotide sequence of a DNA fragment containing the alkA gene and its control region has been determined using a chemical method. Only one open reading frame responsible for 3-methyladenine DNA glycosylase II was found. The hypothetical polypeptide deduced from the DNA sequence, with a molecular weight of 31,400, has an amino-terminal sequence and total amino acid composition identical to that of purified 3-methyladenine DNA glycosylase II. We constructed hybrid plasmids carrying an alkA'-lacZ' fusion, with the proper control region for alkA expression. A hybrid polypeptide with beta-galactosidase activity was formed when lac mutant cells harboring such plasmids were incubated with low doses of N-methyl-N'-nitro-N-nitrosoguanidine or methylmethane sulfonate. Other DNA-damaging agents, such as ethylmethane sulfonate, nalidixic acid, and ultraviolet light did not induce the enzyme activity. The induction was controlled by the ada and adc, but not by the recA and lexA genes.     

Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.     

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.     

Imbalanced Base Excision Repair Increases Spontaneous Mutation and Alkylation Sensitivity in Escherichia coli

Inappropriate expression of 3-methyladenine (3MeA) DNA glycosylases has been shown to have harmful effects on microbial and mammalian cells. To understand the underlying reasons for this phenomenon, we have determined how DNA glycosylase activity and substrate specificity modulate glycosylase effects in Escherichia coli. We compared the effects of two 3MeA DNA glycosylases with very different substrate ranges, namely, the Saccharomyces cerevisiae Mag1 and the E. coli Tag glycosylases. Both glycosylases increased spontaneous mutation, decreased cell viability, and sensitized E. coli to killing by the alkylating agent methyl methanesulfonate. However, Tag had much less harmful effects than Mag1. The difference between the two enzymes' effects may be accounted for by the fact that Tag almost exclusively excises 3MeA lesions, whereas Mag1 excises a broad range of alkylated and other purines. We infer that the DNA lesions responsible for changes in spontaneous mutation, viability, and alkylation sensitivity are abasic sites and secondary lesions resulting from processing abasic sites via the base excision repair pathway.     

AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans.

An endonuclease specific for apurinic/apyrimidinic (AP) sites was identified and purified from extracts of Deinococcus radiodurans. The enzyme is 34.5 kD, has no activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA, and is active in the presence of EDTA. The addition of up to 10 mM Mg2+ or Mn2+ did not affect activity, but higher concentrations were inhibitory. There is no associated exonuclease activity, either in the presence or absence of divalent cation. Optimal reaction conditions were 150 mM NaCl and pH 7.5. A uracil DNA glycosylase was also detected, active in the presence of EDTA, selectively removing uracil from DNA without generating other byproducts. The optimal reaction conditions were 50 mM NaCl and pH 7.5. Implications for base excision repair in D. radiodurans are discussed.