Methylating Agent N-methyl-N'-nitro-N-nitrosoguanidine Research Articles

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

Exonuclease 1 (Exo1) is required for activating response to SN1 DNA methylating agents

SN1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O6 position of guanine (O6meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O6meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the SN1 methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to SN1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.

O6-Methylguanine DNA lesions induce an intra-S-phase arrest from which cells exit into apoptosis governed by early and late multi-pathway signaling network activation

The O(6)-methylguanine (O(6)MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O(6)MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O(6)MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O(6)MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O(6)MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.

Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1.

In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutS alpha) and hMSH2/hMSH3 (hMutS beta) are the mismatch recognition factors of the mismatch repair pathway. hMutS alpha has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutS alpha and hMutL alpha. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutS alpha binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutS alpha. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.

Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles

The DNA adduct O6-methylguanine (O6MeG) induced by environmental genotoxins and anticancer drugs is a highly mutagenic, genotoxic and apoptotic lesion. Apoptosis induced by O6MeG requires mismatch repair (MMR) and proliferation. Models of O6MeG-triggered cell death postulate that O6MeG/T mispairs activate MMR giving rise to either direct genotoxic signaling or secondary lesions that trigger apoptotic signaling in the 2nd replication cycle. To test these hypotheses, we used a highly synchronized cell system competent and deficient for the repair of O6MeG adducts, which were induced by the SN1 methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). We show that DNA double-strand breaks (DSBs) are formed in response to O6MeG at high level in the 2nd S/G2-phase of the cell cycle. This is accompanied by ATR and Chk1 phosphorylation, G2/M arrest and late caspase activation. Although cells undergo apoptosis out of the 2nd G2/M-phase, the majority of them recovers and undergoes apoptosis after passing through additional replication cycles. The late apoptotic effects were completely abolished by O6-methylguanine-DNA methyltransferase, indicating that non-repaired O6MeG is carried over into subsequent generations, eliciting there a late apoptotic response. We also demonstrate that with a low, non-toxic dose of MNNG the passage of cells through the 1st and 2nd S-phase is not delayed, although the dose is able to induce excessive sister chromatid exchanges. This suggests that a significant amount of O6MeG can be tolerated by recombination, which is a fast process preventing from S-phase blockage, DSB formation and cell death.

Interplay of DNA Repair Pathways Controls Methylation Damage Toxicity in Saccharomyces cerevisiae

Methylating agents of S(N)1 type are widely used in cancer chemotherapy, but their mode of action is poorly understood. In particular, it is unclear how the primary cytotoxic lesion, O(6)-methylguanine ((Me)G), causes cell death. One hypothesis stipulates that binding of mismatch repair (MMR) proteins to (Me)G/T mispairs arising during DNA replication triggers cell-cycle arrest and cell death. An alternative hypothesis posits that (Me)G cytotoxicity is linked to futile processing of (Me)G-containing base pairs by the MMR system. In this study, we provide compelling genetic evidence in support of the latter hypothesis. Treatment of 4644 deletion mutants of Saccharomyces cerevisiae with the prototypic S(N)1-type methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) identified MMR as the only pathway that sensitizes cells to MNNG. In contrast, homologous recombination (HR), postreplicative repair, DNA helicases, and chromatin maintenance factors protect yeast cells against the cytotoxicity of this chemical. Notably, DNA damage signaling proteins played a protective rather than sensitizing role in the MNNG response. Taken together, this evidence demonstrates that (Me)G-containing lesions in yeast must be processed to be cytotoxic.

Function of Domains of Human O6-Alkylguanine-DNA Alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects from alkylating agents by converting O6-alkylguanine to guanine forming S-methylcysteine in the AGT protein. The crystal structure of human AGT shows clearly the presence of two domains. The N-terminal domain contains a bound zinc atom, and zinc binding confers a mechanistic enhancement to repair activity, but this domain has no known function. The C-terminal domain contains all residues so far implicated in alkyl transfer including the cysteine acceptor site (Cys145), the O6-alkylguanine binding pocket, and a DNA binding domain. We have expressed and purified the two domains of human AGT separately. The C-terminal domain was totally inactive in vitro, but good activity forming S-alkylcysteine at Cys145 was obtained after recombination with the N-terminal domain via a freeze-thawing procedure. This suggests that the N-terminal domain plays a critical structural role in maintaining an active configuration of the C-terminal domain. However, this C-terminal domain alone had activity in protecting against the cytotoxic and mutagenic activity of the methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) when expressed in Escherichia coli cells lacking endogenous AGT, suggesting that other proteins can fulfill this function. Remarkably, the free N-terminal domain of hAGT was able to repair O6-alkylguanine in vitro via alkyl transfer provided that zinc ions were present. The N-terminal domain was also able to produce moderate protection from MNNG when expressed in E. coli. This cryptic Zn2+-dependent DNA repair activity may be relevant to the evolution and function of AGTs.

High Doses of SN1 Type Methylating Agents Activate DNA Damage Signaling Cascades that are Largely Independent of Mismatch Repair

Methylating agents of the SN1 type represent an important class of cancer chemotherapeutics. Efficient killing by clinically-relevant doses of these agents requires cell division and low levels or absence of the repair enzyme methylguanine methyl transferase (MGMT). The process requires also an active mismatch repair (MMR) system, as treatment of cells with the prototypic methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) could be shown to trigger a delayed cell cycle arrest, which was absolutely MMR-dependent. We now show that DNA damage signaling activated by high doses of MNNG is very rapid and largely MMR-independent. However, the MMR system still contributes towards cell killing, as MMR deficiency favors the long-term survival of the cells, albeit to a substantially smaller extent than when low MNNG concentrations are deployed.

Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase.

S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.

MSH2 and ATR form a signaling module and regulate two branches of the damage response to DNA methylation.

The mismatch repair proteins function upstream in the DNA damage signaling pathways induced by the DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We report that MSH2 (MutS homolog 2) protein interacts with the ATR (ATM- and Rad3-related) kinase to form a signaling module and regulate the phosphorylation of Chk1 and SMC1 (structure maintenance of chromosome 1). We found that phosphorylation of Chk1 by ATR also requires checkpoint proteins Rad17 and replication protein A. In contrast, phosphorylation of SMC1 by ATR is independent of Rad17 and replication protein A, suggesting that the signaling pathway leading to SMC1 phosphorylation is distinct from that mediated by the checkpoint proteins. In addition, both MSH2 and Rad17 are required for the activation of the S-phase checkpoint to suppress DNA synthesis in response to MNNG, and phosphorylation of SMC1 is required for cellular survival. These data support a model in which MSH2 and ATR function upstream to regulate two branches of the response pathway to DNA damage caused by MNNG.

The base excision repair enzyme MED1 mediates DNA damage response to antitumor drugs and is associated with mismatch repair system integrity.

Cytotoxicity of methylating agents is caused mostly by methylation of the O6 position of guanine in DNA to form O6-methylguanine (O6-meG). O6-meG can direct misincorporation of thymine during replication, generating O6-meG:T mismatches. Recognition of these mispairs by the mismatch repair (MMR) system leads to cell cycle arrest and apoptosis. MMR also modulates sensitivity to other antitumor drugs. The base excision repair (BER) enzyme MED1 (also known as MBD4) interacts with the MMR protein MLH1. MED1 was found to exhibit thymine glycosylase activity on O6-meG:T mismatches. To examine the biological significance of this activity, we generated mice with targeted inactivation of the Med1 gene and prepared mouse embryonic fibroblasts (MEF) with different Med1 genotype. Unlike wild-type and heterozygous cultures, Med1-/- MEF failed to undergo G2-M cell cycle arrest and apoptosis upon treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Similar results were obtained with platinum compounds' 5-fluorouracil and irinotecan. As is the case with MMR-defective cells, resistance of Med1-/- MEF to MNNG was due to a tolerance mechanism because DNA damage accumulated but did not elicit checkpoint activation. Interestingly, steady state amounts of several MMR proteins are reduced in Med1-/- MEF, in comparison with Med1+/+ and Med1+/- MEF. We conclude that MED1 has an additional role in DNA damage response to antitumor agents and is associated with integrity of the MMR system. MED1 defects (much like MMR defects) may impair cell cycle arrest and apoptosis induced by DNA damage.

Methylation-induced G(2)/M arrest requires a full complement of the mismatch repair protein hMLH1.

The mismatch repair (MMR) gene hMLH1 is mutated in approximately 50% of hereditary non-polyposis colon cancers and transcriptionally silenced in approximately 25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR-proficient and arrested at the G(2)/M cell cycle checkpoint following treatment with the DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR-proficient cells, activation of the MNNG-induced G(2)/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.

Carcinogen-specific induction of genetic instability.

It has been proposed recently that the type of genetic instability in cancer cells reflects the selection pressures exerted by specific carcinogens. We have tested this hypothesis by treating immortal, genetically stable human cells with representative carcinogens. We found that cells resistant to the bulky-adduct-forming agent 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) exhibited a chromosomal instability (CIN), whereas cells resistant to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exhibited a microsatellite instability (MIN) associated with mismatch repair defects. Conversely, we found that cells purposely made into CIN cells are resistant to PhIP, whereas MIN cells are resistant to MNNG. These data demonstrate that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability and vice versa.

Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase.

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.

Common fragile site at XQ21 in Chinese hamster and its implications for industrial clastogenicity testing

SN1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O6 position of guanine (O6meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O6meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the SN1 methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to SN1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.

Contribution of O6‐methylguanine‐DNA methyltransferase to monofunctional alkylating‐agent resistance in human brain tumor—derived cell lines

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and glioma-derived cell lines differ in sensitivity to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6-benzylguanine (O6-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD10 among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial.

X-Ray Induction of O 6 -Alkylguanine-DNA Alkyltransferase Protects against Some of the Biological Effects of N-Methyl-N prime -Nitro-N-Nitrosoguanidine in C3H 10T1/2 Cells

We have shown previously that the repair of O6-methylguanine can be induced in murine fibroblasts (C3H 10T1/2 cells) by exposure to X rays. The magnitude of the response is less, however, than is observed in the well-characterized adaptive response of various prokaryotes to methylating agents. To determine whether the induction of O6-alkylguanine-DNA alkyltransferase in C3H 10T1/2 cells is sufficient for protection against the genotoxic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cells were challenged with MNNG after alkyltransferase induction by 1.5 Gy X rays and assayed for cytotoxicity, mutagenicity, and neoplastic transformation. Preirradiated cells were significantly more resistant to the mutagenic effects of MNNG as scored by formation of ouabain-resistant colonies. The protective effect was greatest in cells challenged with a low dose (0.2 or 0.4 micrograms/ml) of MNNG. Protection against neoplastic transformation by MNNG was also observed, although the protective effect in this case was significant only in cells treated with a high dose (1.0 micrograms/ml) of MNNG. In cells that were preirradiated, there was no reduction in the cytotoxicity caused by MNNG or the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These data indicate that alkyltransferase induction in C3H 10T1/2 cells is sufficient to protect cells against some of the genotoxic effects of the alkylating agent MNNG. The data also suggest that formation of O6-alkylguanine may not be the only means by which alkylating agents can transform C3H 10T1/2 cells.

Poly ADP-ribosylation and DNA strand breakage in SV40 minichromosomes.

Poly ADP-ribosylation and DNA strand breakage in response to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were studied on SV40 minichromosomes in SV40-infected, permeabilized CV-1 monkey cells. After an initial sharp increase in poly ADP-ribosylation, strand breakage and poly ADPR increased proportionately with increasing dose of MNNG. This suggests a cause-effect relationship between the two reactions. The major poly ADP-ribose acceptor of minichromosomes was core histone H2B. In contrast, H2B, H2A, H1 and protein A24 were poly ADP-ribosylated in the nuclear chromatin of the same cells.

Mismatch correction at O6-methylguanine residues in E. coli DNA.

Escherichia coli has a correction system which removes mismatched bases from DNA1. Mutants (dam) which lack the major DNA adenine methylase2 are hypersensitive to the effects of base analogue mutagens such as 2-aminopurine3 and appear to be defective in mismatch correction. Phenotypic revertants of dam mutants to base analogue resistance include second site mutations in mutL or mutS genes4, which are also part of the correction system. We report here that E. coli dam mutants are also sensitive to the DNA methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which introduces O6-methylguanine (m6G) into the DNA. This sensitivity, however, was not observed using methylating agents which generate only low amounts of this alkylated base. Furthermore, the introduction of either a mutL or a mutS mutation into dam strains abolished the sensitivity to MNNG. These results suggest that mismatch correction occurs at m6G residues in DNA. These lesions miscode in DNA polymerase I-mediated DNA synthesis in vitro5 and are known to be mutagenic in vivo6. Nevertheless, it appears that mismatch correction at m6G residues in DNA does not lead to reduced induction of mutation by MNNG.

An adaptive response of E. coli to low levels of alkylating agent: Comparison with previously characterised DNA repair pathways

We have described previously an inducible response in Escherichia coli which occurs during growth on low levels of the methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and which enables cells both to survive better and to be less mutated by a subsequent challenge dose of MNNG than control cultures (Samson and Cairns, 1977). We show here that this response is distinct from previously characterised pathways of DNA repair, and particularly from the SOS response, which is another inducible effect resulting from DNA damage. An examination of the cross-reactivity of this response with other mutagens has shown that it is a generalised mechanism affecting alkylation damage to DNA. It cannot, however, be induced by UV or the UV-mimetic mutagen, 4-nitroquinoline 1-oxide, nor act on lesions put into DNA by those mutagens.