DNA Methylation Damage Research Articles

Mismatch repair and differential sensitivity of mouse and human cells to methylating agents.

The long-patch mismatch repair pathway contributes to the cytotoxic effect of methylating agents and loss of this pathway confers tolerance to DNA methylation damage. Two methylation-tolerant mouse cell lines were identified and were shown to be defective in the MSH2 protein by in vitro mismatch repair assay. A normal copy of the human MSH2 gene, introduced by transfer of human chromosome 2, reversed the methylation tolerance. These mismatch repair defective mouse cells together with a fibroblast cell line derived from an MSH2-/- mouse, were all as resistant to N-methyl-N-nitrosourea as repair-defective human cells. Although long-patch mismatch repair-defective human cells were 50- to 100-fold more resistant to methylating agents than repair-proficient cells, loss of the same pathway from mouse cells conferred only a 3-fold increase. This discrepancy was accounted for by the intrinsic N-methyl-N-nitrosourea resistance of normal or transformed mouse cells compared with human cells. The >20-fold differential resistance between mouse and human cells could not be explained by the levels of either DNA methylation damage or the repair enzyme O6-methylguanine-DNA methyltransferase. The resistance of mouse cells to N-methyl-N-nitrosourea was selective and no cross-resistance to unrelated DNA damaging agents was observed. Pathways of apoptosis were apparently intact and functional after exposure to either N-methyl-N-nitrosourea or ultraviolet light. Extracts of mouse cells were found to perform 2-fold less long-patch mismatch repair. The reduced level of mismatch repair may contribute to their lack of sensitivity to DNA methylation damage.

Synergism between yeast nucleotide and base excision repair pathways in the protection against DNA methylation damage.

The treatment of cells with simple DNA methylating agents such as methyl methanesulfonate (MMS) results in genotoxic lesions, including 3-methyladenine which blocks DNA replication. All the organisms studied to date contain an alkylation-specific base excision repair pathway. In the yeast Saccharomyces cerevisiae, the base excision repair pathway is initiated by a Mag1 3-methyladenine DNA glycosylase that removes the damaged base, followed by the Apn1 apurinic/apyrimidinic endonuclease which cleaves the DNA strand at the abasic site for subsequent repair and synthesis. Several nucleotide excision repair pathway mutants display only slightly increased sensitivity to killing by MMS, indicating that nucleotide excision repair per se does not play a major role in the repair of DNA methylation damage. However, mag1 and apn1 mutants that are also defective in nucleotide excision repair are extremely sensitive to MMS-induced killing and the effects are synergistic. These observations suggest that nucleotide excision repair and alkylation-specific base excision repair provide alternative pathways for the repair of DNA methylation damage. In addition to their role in nucleotide excision repair, Rad1 and Rad10 form a complex that is involved in recombination repair. It was found that the apn1 rad1 and apn1 rad10 double mutants have a growth defect and are significantly more sensitive to MMS killing than apn1 rad2 and apn1 rad4 double mutants in a gradient plate assay. Furthermore, the apn1 rad1 double mutant increased both the spontaneous and MMS-induced mutation frequency. Thus, the recombination repair defects of rad1 and rad10 may confer an additional synergistic effect when combined with the apn1 mutation.

Methyl methanesulfonate-induced hprt mutation spectra in the Chinese hamster cell line CHO9 and its xrcc1-deficient derivative EM-C11

The Chinese hamster cell mutant EM-C11, which is hypersensitive to the cell killing effects of alkylating agents compared to its parental line CHO9, has been used to study the impact of base excision repair on the mutagenic effects of DNA methylation damage. This cell line has a defect in the xrcc1 gene. XRCC1 can interact with DNA polymerase-β, thereby suppressing strand displacement, and DNA ligase III, both of which have been implicated in base excision repair. XRCC1 may, therefore, allow efficient ligation of single-strand breaks generated during base excision repair. Both EM-C11 and CHO9 cells were treated with methyl methanesulfonate (MMS), a DNA-methylating agent reacting predominantly with nitrogen atoms generating adducts which are substrates for the base excision repair pathway. EM-C11 cells are much more sensitive to the cytotoxic effects of MMS than CHO9: for EM-C11, the dose of MMS inducing 10% survival is 6-fold lower than that for CHO9. In contrast, mutation induction at the hprt locus following MMS is similar in EM-C11 and CHO9. Molecular analysis of hprt gene mutations showed that although the largest class of hprt mutations, both in EM-C11 and CHO9 cells, consisted of GC>AT transitions, most likely caused by O 6-methylguanine, the size of this class was smaller in EM-C11. The fraction of deletion mutants in EM-C11, however, was twice as large as that found in CHO9 cells. These results suggest that reduced ligation efficiency of single-strand breaks generated during base excision repair, as result of a defect in XRCC1, may lead to the formation of deletions.

Reversal of methylation tolerance by transfer of human chromosome 2

Human cell lines resistant to N-methyl-N-nitrosourea (MNU) were previously assigned to two complementation groups. Members of group I are defective in mismatch correction [S. Ceccotti, G. Aquilina, P. Macpherson, M. Yamada, P. Karran, M. Bignami, Processing of O6-methylguanine by mismatch correction in human cell extracts. Current Biol. 6 (1996) 1528–1531]. To identify the mechanism responsible for the less pronounced phenotype of the second complementation group, we characterized the persistence of MNU-induced O6-methylguanine (O6-meGua) and mutation induction at the hypoxanthine guanine phosphoribosyl-transferase (HPRT) locus. Group II clones are unable to repair the premutagenic base O6-meGua and are as mutable by MNU as group I clones and the parental HeLaMR cells. MNU-induced SCE were undetectable in group I clones and drastically reduced in group II in comparison with the parental cells. These observations are consistent with a defective processing of DNA methylation damage by members of both groups. Group II clones exhibit a moderate spontaneous mutator phenotype at the HPRT gene but significant instability at mononucleotide repeat microsatellites. Introduction of a single human chromosome 2 (but not of chromosome 3 or 7) into group II cells partially reverts both MNU resistance and the increased spontaneous mutation rate. The properties of group II variants are consistent with methylation tolerance and a partially defective mismatch repair. We propose that members of group II are defective in the chromosome 2-based mismatch correction gene GTBP/hMSH6.

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, delta and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae.

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase delta, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase delta and a deletion in the REV3 gene had a complete repair defect, both at permissive (23 degrees C) and restrictive (38 degrees C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase epsilon, nor deletion in the DNA polymerase IV (beta70) gene, caused defective repair. The data obtained suggest that DNA polymerases delta and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis.

The repair of DNA methylation damage in Saccharomyces cerevisiae.

The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways.

Drug-related killings: a case of mistaken identity.

Abortive attempts at DNA repair can contribute to the effects of DNA damage inflicted by cytotoxic drugs. DNA methylation damage, 6-thioguanine and cisplatin adducts all owe their cytotoxicity in part to the intervention of DNA mismatch repair.

RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes.

The ada and aidB genes are part of the adaptive response to DNA methylation damage in Escherichia coli. Transcription of the ada and the aidB genes is triggered by binding of the methylated Ada protein (meAda) to a specific sequence located 40-60 base pairs upstream of the transcriptional start, which is internal to an A/T-rich region. In this report we demonstrate that the Ada binding site is also a binding site for RNA polymerase. RNA polymerase is able to bind the -40 to -60 region of the ada and the aidB promoters in the absence of meAda, and its binding is mediated by the alpha subunit. This region resembles the UP element of the rrnB P1 promoter in location, sequence and mechanism of interaction with RNA polymerase. We discuss the function of UP-like elements in positively controlled promoters and provide evidence that Ada does not act by enhancing RNA polymerase binding affinity to the promoter region. Instead, Ada stimulates transcription by modifying the nature of the RNA polymerase-promoter interaction, allowing RNA polymerase to recognize the core promoter -35 and -10 elements in addition to the UP-like element.

Streptozotocin-induced genotoxic effects in Chinese hamster cells: The resistant phenotype of V79 cells

The genotoxic effects of the methylating agent streptozotocin (STZ) on Chinese hamster cells CHO-9 and V79 were evaluated. The induction of cell killing, chromosomal aberrations, sister-chromatid exchanges (SCEs) and mutations was analyzed. Comparisons were made with the the STZ aglyconic analogue N-methyl- N-nitrosourea (MNU). V79 cells were found to be more resistant than CHO-9 cells to STZ and MNU killing effects, as well as to the induction of chromosomal aberrations and SCEs; however, V79 and CHO-9 cells appeared to be equally sensitive to the induction of 6-thioguanine resistant mutants by STZ. These results suggest that an error-free mechanisms that tolerates DNA methylation damage confers a resistant phenotype to V79 cells to the genotoxic effects of methylation damage.

Transcriptional Activation of the Escherichia coli Adaptive Response Gene aidB Is Mediated by Binding of Methylated Ada Protein: EVIDENCE FOR A NEW CONSENSUS SEQUENCE FOR Ada-BINDING SITES

The Escherichia coli aidB gene is part of the adaptive response to DNA methylation damage. Genes belonging to the adaptive response are positively regulated by the ada gene; the Ada protein acts as a transcriptional activator when methylated in one of its cysteine residues at position 69. Through DNaseI protection assays, we show that methylated Ada (meAda) is able to bind a DNA sequence between 40 and 60 base pairs upstream of the aidB transcriptional startpoint. Binding of meAda is necessary to activate transcription of the adaptive response genes; accordingly, in vitro transcription of aidB is dependent on the presence of meAda. Unmethylated Ada protein shows no protection against DNaseI digestion in the aidB promoter region nor does it promote aidB in vitro transcription. The aidB Ada-binding site shows only weak homology to the proposed consensus sequences for Ada-binding sites in E. coli (AAANNAA and AAAGCGCA) but shares a higher degree of similarity with the Ada-binding regions from other bacterial species, such as Salmonella typhimurium and Bacillus subtilis. Based on the comparison of five different Ada-dependent promoter regions, we suggest that a possible recognition sequence for meAda might be AATnnnnnnG-CAA. Higher concentrations of Ada are required for the binding of aidB than for the ada promoter, suggesting lower affinity of the protein for the aidB Ada-binding site. Common features in the Ada-binding regions of ada and aidB are a high A/T content, the presence of an inverted repeat structure, and their position relative to the transcriptional start site. We propose that these elements, in addition to the proposed recognition sequence, are important for binding of the Ada protein.

DNA polymerase delta is required for base excision repair of DNA methylation damage in Saccharomyces cerevisiae.

We present evidence that DNA polymerase delta of Saccharomyces cerevisiae, an enzyme that is essential for viability and chromosomal replication, is also required for base excision repair of exogenous DNA methylation damage. The large catalytic subunit of DNA polymerase delta is encoded by the CDC2(POL3) gene. We find that the mutant allele cdc2-2 confers sensitivity to killing by methyl methanesulfonate (MMS) but allows wild-type levels of UV survival. MMS survival of haploid cdc2-2 strains is lower than wild type at the permissive growth temperature of 20 degrees C. Survival is further decreased relative to wild type by treatment with MMS at 36 degrees C, a nonpermissive temperature for growth of mutant cells. A second DNA polymerase delta allele, cdc2-1, also confers a temperature-sensitive defect in MMS survival while allowing nearly wild-type levels of UV survival. These observations provide an in vivo genetic demonstration that a specific eukaryotic DNA polymerase is required for survival of exogenous methylation damage. MMS sensitivity of a cdc2-2 mutant at 20 degrees C is complemented by expression of mammalian DNA polymerase beta, an enzyme that fills single-strand gaps in duplex DNA in vitro and whose only known catalytic activity is polymerization of deoxyribonucleotides. We conclude, therefore, that the MMS survival deficit in cdc2-2 cells is caused by failure of mutant DNA polymerase delta to fill single-strand gaps arising in base excision repair of methylation damage. We discuss our results in light of current concepts of the physiologic roles of DNA polymerases delta and epsilon in DNA replication and repair.

O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY IN TISSUES OF MICE TREATED WITH ANTISCHISTOSOMAL AGENTS

The activity of O-6-alkylguanine-DNA alkyl transferase (ATase), the enzyme responsible for repairing promutagenic methylation damage in DNA, was measured at various time intervals in tissue extracts of mice administered (in vivo) a single therapeutic dose of the antischistosomal agents hycanthone, oxaminiquine and metrifonate. In control animals, liver contained the highest levels of ATase activity (15.8+/-1.8 fmole ATase/mu g DNA) followed by spleen (11.0+/-1.7 fmole/mu g DNA), intestine (2.3+/-0.3 fmole/mu g DNA) and bladder (0.22+/-0.04 fmole/mu g DNA). With hycanthone, ATase activity was reduced by 6 and 24 h post treatment to 74% and 27% below the control value, respectively. Bladder exhibited a 25% inactivation in the ATase level at 6 h time point. Spleen and bladder did not show any alteration in the ATase activity. In animals administered oxaminiquine, liver and bladder had a near identical pattern to that observed for hycanthone. Spleen and intestine, however, revealed activation in ATase by 50% and 42%, respectively after 6 h of treatment. This activation was also observed in the bladder of metrifonate-treated mice. In a previous study (Badawi et al, Cancer Lett 75: 167, 1993), DNA-alkylation damage (O-6-methyldeoxyguanosine; O-6-MedG) was evaluated in these tissues and there was an inverse correlation between the levels of methylation damage and ATase activity in liver (r=-0.85, p<0.01), intestine (r=-0.62, p<0.01) and bladder (r=-0.59, p<0.05).

DNA mismatch binding and incision at modified guanine bases by extracts of mammalian cells: implications for tolerance to DNA methylation damage.

Two activities involved in separate pathways for correcting G.T mispairs in DNA have been assayed on duplex substrates containing modified guanine bases. The first, the G.T mismatch incision activity, is specifically involved in short-patch repair of mispairs arising via deamination of 5-methylcytosine. The second activity can be detected by its ability to bind to G.T mispairs and may initiate correction by a long-patch mechanism. 6-Thioguanine and O6-methylguanine paired with thymine were efficiently incised by cell extracts if the modified guanine was in a CpG dinucleotide. Incision was not observed when either purine was paired with cytosine. Extracts of cells that are tolerant both to methylation damage and to 6-thioguanine in DNA also incised 6-thioguanine.T and O6-methylguanine.T base pairs. The data suggest that this activity is unlikely to contribute significantly to the biological effects of O6-methylguanine in DNA. A defect in this pathway is therefore unlikely to explain the cross-resistance of tolerant cells to the two base analogs in DNA. In binding assays, 6-thioguanine.T base pairs were recognized efficiently and to an equivalent extent by the same protein complex as G.T mispairs. O6-Methylguanine.T base pairs were also recognized but with reduced efficiency. No binding was observed to 6-thioguanine.C or O6-methylguanine.C base pairs. Recognition by the binding complex was essentially independent of the base immediately 5' to the mismatched guanine but was somewhat more efficient if O6-methylguanine was preceded by a purine. Extracts of two tolerant lines with a known defect in G.T mismatch binding failed to form complexes with substrates containing the modified bases.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA replication arrest and tolerance to DNA methylation damage.

Inhibition of DNA replication by different DNA damaging agents has been investigated in HeLaMR cells and a methylation damage-tolerant variant HeLa5A1. In synchronous HeLaMR and HeLa5A1 cells exposed to N-ethyl-N-nitrosourea or ionizing radiation in mid-G1 phase, DNA synthesis was inhibited in the following S phase. N-methyl-N-nitrosourea-induced replication inhibition in HeLaMR cells was delayed until the second S phase after treatment. In contrast, N-methyl-N-nitrosourea treatment of HeLa5A1 cells affected neither the timing nor the extent of the first or second S phases. Both radiation and chemical treatment inhibited replication of an episomal plasmid and of genomic DNA in unison. Inhibition was observed at levels of DNA damage that did not directly damage the plasmid molecules. Thus, DNA replication inhibition occurs immediately after ionizing radiation or ethylation damage, but methylation damage requires processing through one cell cycle to generate an inhibitory signal. The inhibitory signal appears to act in trans on undamaged DNA. Although methylation-tolerant cells are responsive to inhibition after gamma-irradiation, methylation damage does not produce inhibitory signals to which they respond.

Promutagenic methylation damage in liver DNA of mice infected with Schistosoma mansoni.

Male and female BK-TO mice were infected with different numbers of Schistosoma mansoni cercariae under standard environmental conditions. Promutagenic methylation damage (O6-methyldeoxyguanosine; O6-MedG) was detected in liver DNA, but not in kidney, spleen or bladder DNA of infected animals. It was shown that levels of hepatic O6-MedG increased with increasing intensities of schistosomal infection. Possible mechanisms of action are discussed. These include the activating effects of schistosomes and their products on murine macrophages and subsequent endogenous formation of N-nitroso compounds by the activated macrophages.

Genetic consequences of tolerance to methylation DNA damage in mammalian cells.

We previously characterized a clone of CHO cells, clone B, that displayed tolerance to the cytotoxic effects of N-methylnitrosourea (MNU) and 6-thioguanine (6-TG). To determine whether this phenotype affected the mutagenic response of the cells, MNU-induced mutation to 8-azaadenine resistance (8-AAr) was measured in the parental and clone B cells. Comparable mutation frequencies were found in the two cell lines up to 0.5 mM MNU, while at higher MNU concentrations mutations could be reproducibly measured only in clone B cells. Similar amounts of DNA methylated bases were found in the two cell lines after a 30 min treatment with different concentrations of [3H]MNU and the same linear relationship was observed when mutation induction by MNU was plotted as a function of the amount of O6-methylguanine (O6-MeGua) in DNA, indicating that mutation induction in both cell lines was related to the presence of this methylated base. Fifteen MNU-induced 8-AAr mutants were isolated from each cell line and the sequences of the adenine phosphoribosyltransferase (aprt) mutations determined. The type (in 90% of the cases, GC to AT transitions), the sequence context and the strand localization of the mutations indicated that all mutations were targeted at O6-MeGua in DNA and no difference was found between the two lines. These results are consistent with a mechanism of tolerance of O6-MeGua that does not alter the processing of this methylated base into a mutation. Growth in 6-TG induced point mutations in clone B but not in the parental cells. A model is proposed in which the alkylation tolerant variant is altered in a mismatch correction pathway responsible for the cytotoxicity of the methylated base.

DNA methylation damage in individuals at high risk for stomach cancer

DNA methylation damage in individuals at high risk for stomach cancer

Promutagenic methylation damage in bladder DNA from patients with bladder cancer associated with schistosomiasis and from normal individuals.

Radioimmunoassays (RIAs) have been used to detect the promutagenic lesion O6-methyldeoxyguanosine (O6-MedG) in DNA isolated from the bladder tissues of Egyptian patients presenting with bladder carcinoma and concomitant schistosomiasis (bilharziasis), a parasitic disease known to be associated with the presence of N-nitrosamines in the urine. Alkylation damage was present in the DNA of the majority of the samples (44/46, 96%); 38 samples were of tumour tissue and 8 from uninvolved bladder mucosa. Levels of O6-MedG ranged from undetectable (ND; i.e. less than 0.01 mumol O6-MedG/mol dG) to 0.485 mumol/mol dG with an overall mean of 0.134 +/- 0.10 mumol/mol dG, including the two samples that were below the limit of detection. In contrast, methylation damage was detected in only 4/12 (33%) of the DNA samples from normal bladder tissue of European origin. In these samples levels of O6-MedG ranged from ND to 0.225 mumol/mol dG with an overall mean of 0.046 +/- 0.082 mumol O6-MedG/mol dG. These results confirm that alkylation events can be detected in the DNA of schistosome-infected human bladder tissue. The methylation of uninvolved and tumour tissue DNA to similar extents suggests that the alkylating intermediate may have been present in the urine. These results indicate the need for further investigation to determine whether relationships exist between levels of DNA damage and the prevalence of schistosome infection and/or the extent and type of bacterial infection that frequently accompanies this disease.

Formula omitted]6-alkylguanine-DNA-alkyltransferase activity in relation to the promutagenic methylation damage in bladder DNA from humans predisposed to bladder cancer associated with schistosomiasis : Badawi 1,3, A.F., Cooper 1, D.P., Mostafa 3, M.H., Aboul-Azm 2, T., Margison 1, G.P. and O'Connor 1, P.J. 1CRC Section of Carcinogenesis, Paterson Institute for Cancer Research, Manchester M20 9BX, UK, 2Medical Research Institute, 3Institute of

formula omitted]6-alkylguanine-DNA-alkyltransferase activity in relation to the promutagenic methylation damage in bladder DNA from humans predisposed to bladder cancer associated with schistosomiasis : Badawi 1,3, A.F., Cooper 1, D.P., Mostafa 3, M.H., Aboul-Azm 2, T., Margison 1, G.P. and O'Connor 1, P.J. 1CRC Section of Carcinogenesis, Paterson Institute for Cancer Research, Manchester M20 9BX, UK, 2Medical Research Institute, 3Institute of

The induction of chromosomal damage in rat hepatocytes and lymphocytes II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine, dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O 6-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.