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All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O 6 -alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O 6 -methylguanine ( O 6 MeG) and O 6 -chloroethylguanine ( O 6 CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSα branch of the DNA mismatch repair pathway (but not the MutSβ branch) is absolutely required for signaling the initiation of apoptosis in response to O 6 MeGs and is partially required for signaling apoptosis in response to O 6 CEGs. Further, O 6 MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutSα-dependent. Despite this, MutSα-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.

We have generated mice deficient in O6-methylguanine DNA methyltransferase activity encoded by the murine Mgmt gene using homologous recombination to delete the region encoding the Mgmt active site cysteine. Tissues from Mgmt null mice displayed very low O6-methylguanine DNA methyltransferase activity, suggesting that Mgmt constitutes the major, if not the only, O6-methylguanine DNA methyltransferase. Primary mouse embryo fibroblasts and bone marrow cells from Mgmt -/- mice were significantly more sensitive to the toxic effects of the chemotherapeutic alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin and temozolomide than those from Mgmt wild-type mice. As expected, Mgmt-deficient fibroblasts and bone marrow cells were not sensitive to UV light or to the crosslinking agent mitomycin C. In addition, the 50% lethal doses for Mgmt -/- mice were 2- to 10-fold lower than those for Mgmt +/+ mice for 1,3-bis(2chloroethyl)-1-nitrosourea, N-methyl-N-nitrosourea and streptozotocin; similar 50% lethal doses were observed for mitomycin C. Necropsies of both wild-type and Mgmt -/mice following drug treatment revealed histological evidence of significant ablation of hematopoietic tissues, but such ablation occurred at much lower doses for the Mgmt -/- mice. These results demonstrate the critical importance of O6-methylguanine DNA methyltransferase in protecting cells and animals against the toxic effects of alkylating agents used for cancer chemotherapy.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vλ1 light chain genes from naive and memory B cells in DNA repair–deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand–break repair, and AAG-mediated base excision repair.

AlkG lesions can trigger such programmed cell death.Alkylating agents are commonly used for cancer chemotherapybecause they are highly cytotoxic, and it now appears that atleast some of that cytotoxicity can be accounted for byapoptosis. It is generally assumed that DNA damage constitutesthe primary signal for the induction of apoptosis in responseto DNA damaging agents, but it is important to note that theseagents also damage proteins, lipids, RNA and other cellularcomponents. It is currently unclear exactly which cellulartargets are responsible for triggering alkylation-inducedapoptosis. Indeed, apoptosis in response to a variety of toxicagents can be induced in the absence of a nucleus (1,2) andradiation-induced apoptosis in non-thymic tissues requiresactivation of the ceramide signaling pathway at the cellmembrane (3,4). Similarly, UV-induced responses are initiated,at least in part, by signals generated at the plasma membrane(5). Alkylating agents produce a spectrum of DNA damagecomprising over a dozen DNA lesions. Among these, the O

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic ‘barcode’, were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput ‘barcode’ sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised., Unilever (Firm), National Cancer Institute (U.S.) (R01-CA055042 (now R01-ES022872)), Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant NIEHS P30-ES002109)

Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself. The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum. Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E. coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells. The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways. In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms.

We have begun to develop the fission yeast, Schizosaccharomyces pombe, as a eukaryotic model for cellular defenses against alkylating agents. Here we describe the cloning and characterization of a cDNA, designated mag1, encoding a S. pombe 3-methyladenine (3MeA) DNA glycosylase. 3MeA DNA glycosylases in Escherichia coli are encoded by alkA and tag. S. pombe mag1 was cloned by its ability to reverse the alkylation-sensitive phenotype of an alkA tag E. coli double mutant. The expression of S. pombe magl in E. coli confers partial resistance to alkylating agents that produce methyl, ethyl and propyl lesions, and Magl production produces 3MeA DNA glycosylase activity. In contrast to the E. coli alkA and Saccharomyces cerevisiae MAG genes, expression of S. pombe magl was not appreciably induced by alkylating agents. The magl cDNA encodes a protein of 228 amino acids (aa) that shares similarity with 3MeA DNA glycosylases from E. coli (AlkA), Bacillus subtilis (BsAlkA) and S. cerevisiae (MAG). A consensus sequence of 9 aa common to these microbial 3MeA DNA glycosylases is discussed.

DNA mismatch repair defects in certain cell types confer resistance to the cytotoxic effects of alkylating agents, suggesting that a normally functioning DNA mismatch repair pathway can actually mediate alkylation-induced cell death. In eukaryotic cells this phenomenon is only observed in cells lacking adequate DNA methyltransferase for the repair of O 6 -methylguanine (O 6 MeG) DNA lesions. It has been proposed that O 6 MeG may act as a substrate for DNA mismatch repair when paired with cytosine and when mispaired with thymine and that repeated futile DNA mismatch repair at O 6 MeG DNA lesions is cytotoxic. Here we show that the Escherichia coli MutS DNA mismatch repair binding protein does indeed bind specifically to O 6 MeG DNA lesions. In contrast, MutS does not bind DNA containing another O-alkylated base, namely O 4 -methylthymine, or another kind of modified guanine, namely 8-oxoguanine. These results provide direct biochemical evidence for the involvement of DNA mismatch repair in specifically processing O 6 MeG DNA lesions.

In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.

Methods to analyze the intrinsic physical properties of cells – for example, size, density, rigidity, or electrical properties – are an active area of interest in the microfluidics community. Although the physical properties of cells are determined at a fundamental level by gene expression, the relationship between the two remains exceptionally complex and poorly characterized, limiting the adoption of intrinsic separation technologies. To improve our current understanding of how a cell's genotype maps to a measurable physical characteristic and quantitatively investigate the potential of using these characteristics as biomarkers, we have developed a novel screen that combines microfluidic cell sorting with high-throughput sequencing and the haploid yeast deletion library to identify genes whose functions modulate one such characteristic – intrinsic electrical properties. Using this screen, we are able to establish a high-content electrical profile of the haploid yeast gene deletion strains. We find that individual genetic deletions can appreciably alter the electrical properties of cells, affecting [approximately] 10% of the 4432 gene deletion strains screened. Additionally, we find that gene deletions affecting electrical properties in specific ways (i.e. increasing or decreasing effective conductivity at higher or lower electric field frequencies) are strongly associated with an enriched subset of fundamental biological processes that can be traced to specific pathways and complexes. The screening approach demonstrated here and the attendant results are immediately applicable to the intrinsic separations community., Singapore-MIT Alliance, National Science Foundation (U.S.) (NSF IDBR grant DBI-0852654), National Institutes of Health (U.S.) (NIH grant EB005753)

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

It has long been known that cells repair chemically or physically damaged DNA. But the discovery that damaged RNA can also be repaired may come as a surprise. What's more, some of the same enzymes are involved.

The Samson lab has been developing potentially high-throughput methods for measuring DNA repair capacity (DRC) in human cells. These approaches are modeled on the pioneering work of the late Professor Lawrence Grossman and rely on the transfection of plasmids containing specific types of DNA damage and then monitoring the repair of that damage by the expression of a reporter gene that is poorly expressed in the absence of repair. One of our goals is to simultaneously measure DRC for a variety of DNA repair pathways using a multiplexed approach and a collection of fluorescent proteins of different color. So far we have developed methods for measuring relative levels of nucleotide excision repair, homologous recombination, DNA mismatch repair, and MGMT mediated repair. We are currently extending this approach to encompass other DNA repair pathways. Measuring DNA repair capacity in various human cell types may help predict inter-individual differences in the ability of people to respond upon exposure to environmental agents and thus predict their cancer susceptibility. Moreover, knowing the DNA repair capacity of tumor cells for different types of DNA damage could help in choosing more effective chemotherapy regimens. Citation Format: Leona D. Samson. Developing improved methods to measure human DNA repair capacity [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr IA17.

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O6-methyldeoxyguanosine lesions, O6-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O6-mG DNA methyltransferase (MGMT) showed elevated O6-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer's disease., National Institute of Environmental Health Sciences (ES11384 ), National Institute of Environmental Health Sciences (ES11399), National Institute of Environmental Health Sciences (ES011387), National Institute of Environmental Health Sciences (ES07033)

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea. Extracts of E. coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner. The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas.

Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations.

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.

Escherichia coli expresses two DNA repair methyltransferases (MTases) that repair the mutagenic O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT) DNA lesions; one is the product of the inducible ada gene, and here we confirm that the other is the product of the constitutive ogt gene. We have generated various ogt disruption mutants. Double mutants (ada ogt) do not express any O6MeG/O4MeT DNA MTases, indicating that Ada and Ogt are probably the only two O6MeG/O4MeT DNA MTases in E. coli. ogt mutants were more sensitive to alkylation-induced mutation, and mutants arose linearly with dose, unlike ogt+ cells, which had a threshold dose below which no mutants accumulated; this ogt(+)-dependent threshold was seen in both ada+ and ada strains. ogt mutants were also more sensitive to alkylation-induced killing (in an ada background), and overexpression of the Ogt MTase from a plasmid provided ada, but not ada+, cells with increased resistance to killing by alkylating agents. The induction of the adaptive response was normal in ogt mutants. We infer from these results that the Ogt MTase prevents mutagenesis by low levels of alkylating agents and that, in ada cells, the Ogt MTase also protects cells from killing by alkylating agents. We also found that ada ogt E. coli had a higher rate of spontaneous mutation than wild-type, ada, and ogt cells and that this increased mutation occurred in nondividing cells. We infer that there is an endogenous source of O6MeG or O4MeT DNA damage in E. coli that is prevalent in nondividing cells.

Alkylating agents represent one of the major classes of toxic compounds found in our environment. Most of them are potent mutagens and carcinogens (1), and ironically, they are commonly used for the chemotherapeutic treatment of cancer patients. Alkylating agents igtteract with and damage DNA, which can result in mutation and cell death (2). Our goal is to understand at the molecular level how eukaryotic cells respond to DNA alkylation damage, and how they avoid mutation or killing induced by monofunctional alkylating agents such as methylnitrosoguanidine (MNNG) and methyl methanesulfonate (MMS). We have recently taken two approaches to studying the response of eukaryotic cells to DNA alkylation: (1) expressing bacterial DNA repair genes in eukaryotic cells in order to study the biological consequences of various DNA lesions; (2) cloning eukaryotic DNA repair genes by cross species functional complementation in alkylation-sensitive bacteria. Both strategies are based on our understanding of the DNA alkylation repair mechanisms in E. coli.

Yeast strains lacking Anc1, a member of the YEATS protein family, are sensitive to several DNA damaging agents. The YEATS family includes two human genes that are common fusion partners with MLL in human acute leukemias. Anc1 is a member of seven multi-protein complexes involved in transcription, and the damage sensitivity observed in anc1Delta cells is mirrored in strains deleted for some other non-essential members of several of these complexes. Here we show that ANC1 is in the same epistasis group as SRS2 and RAD5, members of the postreplication repair (PRR) pathway, but has additive or synergistic interactions with several other members of this pathway. Although PRR is traditionally divided into an "error-prone" and an "error-free" branch, ANC1 is not epistatic with all members of either established branch, and instead defines a new error-free branch of the PRR pathway. Like several genes involved in PRR, an intact ANC1 gene significantly suppresses spontaneous mutation rates, including the expansion of (CAG)(25) repeats. Anc1's role in the PRR pathway, as well as its role in suppressing triplet repeats, point to a possible mechanism for a protein of potential medical interest.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.

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.

The in vivo removal of three different O-alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for O6-methylguanine, O6-ethylguanine and O4-ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, O6-methylguanine, O6-ethylguanine and O4-ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild-type E. coli the adaptive response began to contribute to O6-methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of O6-ethylguanine and O4-ethylthymine in wild-type E. coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three O-alkylated lesions was virtually absent in ada- uvr- bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of O-alkylated bases was compromised by an ada- or by a uvr- mutation, the bacteria became more sensitive to alkylation induced killing and mutation.

Mer- human cells, which lack O6-methylguanine DNA methyltransferase activity, are extremely sensitive to alkylation induced killing, mutation and sister chromatid exchange. We have analyzed a Mer+, a Mer-, and a Mer- revertant HeLa cell line and found that the methyltransferase deficiency correlates with increased levels of mutation and sister chromatid exchange, but does not correlate with increased killing of Mer- HeLa cells by alkylating agents. Furthermore, we show that HeLa Mer- cells repair N-3-methylguanine and N-3-methyladenine just as efficiently as HeLa Mer+ cells.

In an attempt to characterise which gene products may be involved in the repair system induced in E. coli by growth on low levels of alkylating agent (the adaptive response) we have analysed mutants deficient in other known pathways of DNA repair for the ability to adapt to MNNG. Adaptive resistance to the killing effects of MNNG seems to require a functional DNA polymerase I whereas resistance to the mutagenic effects can occur in polymerase I deficient strains; similarly killing adaptation could not be observed in a dam3 mutant, which was nonetheless able to show mutational adaptation. These results suggest that these two parts of the adaptive response must, at least to some extent, be separable. Both adaptive responses can be seen in the absence of uvrD + uvrE +-dependent mismatch repair, DNA polymerase II activity, or recF-mediated recombination and they are not affected by decreased levels of adenyl cyclase. The data presented support our earlier conclusion that adaptive resistance to the killing and mutagenic effect of MNNG is the result of previously uncharacterised repair pathways.

If eukaryotic genes could protect bacteria with defects in DNA repair, this effect could be exploited for the isolation of eukaryotic DNA repair genes. We have thus cloned a DNA repair gene from Saccharomyces cerevisiae that directs the synthesis of a DNA glycosylase that specifically releases 3-methyladenine from alkylated DNA and in so doing protects alkylation-sensitive Escherichia coli from killing by methylating agents. The cloned yeast gene was then used to generate a mutant strain of S. cerevisiae that carries a defect in the glycosylase gene and is extremely sensitive to DNA methylation. This approach may allow the isolation of a large number of eukaryotic DNA repair genes.

Protein kinases control cellular decision processes by phosphorylating specific substrates. Proteome-wide mapping has identified thousands of in vivo phosphorylation sites. However, systematically resolving which kinase targets each site is presently infeasible, due to the limited specificity of consensus motifs and the potential influence of contextual factors, such as protein scaffolds, localisation and expression, on cellular substrate specificity. We have therefore developed a computational method, NetworKIN, that augments motifs with context for kinases and phosphoproteins. This can pinpoint individual kinases responsible for specific in vivo phosphorylation events and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. We show that context provides 60–80% of the computational capability to assign in vivo substrate specificity. Applying this approach to a DNA damage signalling network, we extend its cell-cycle regulation by showing that 53BP1 is a CDK1 substrate, show that Rad50 is phosphorylated by ATM kinase under genotoxic stress, and suggest novel roles of ATM in apoptosis. Finally, we present a scalable strategy to validate our predictions and use it to support the prediction that BCLAF1 is a GSK3 substrate.

When Escherichia coli is chronically exposed to very low, nontoxic doses of a monofunctional alkylating agent (notably N-methyl-N′-nitro-nitrosoguanidine, MNNG), the adaptive DNA repair pathway is induced which enables the bacteria to resist the killing and mutagenic effects of further alkylation damage1–3. Mutation resistance in adapted bacteria is achieved, at least partly, by a greatly increased capacity of the cells to eliminate the minor DNA alkylation product O6-methyl-guanine4–6, which has been strongly implicated as premu-tagenic7,8 and precarcinogenic9. We now show that the chronic treatment of a Chinese hamster ovary (CHO) and a SV40-transformed human skin fibroblast (GM637) cell line with non-toxic levels of MNNG renders the cells resistant to the induction of sister chromatid exchange (SCE) by further alkylation damage. CHO cells also become resistant to killing (GM637 cells have not yet been tested). Having ruled out explanations such as changes in cell cycle distribution, mutagen permeability and mutagen detoxification, we conclude that resistance is probably achieved by the cells becoming more efficient at repairing alleviation damage, analogous to the adaptive response of E. coli1.

The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains.

Mutagenesis by simple alkylating agents is thought to occur by either a lexA+-dependent process called error-prone repair or a lex-independent process often attributed to mispairing during replication. We show here that error-prone repair is responsible for the majority of mutants formed after a large dose of alkylating agent, but it is unlikely that it contributes significantly to mutagenesis during exposure to low concentrations of these chemicals. The mutagenicity of these low doses of alkylating agent is reduced by a repair system constitutively present in lexA+ cells but absent in lexA mutants. This system reduces mutagenesis until a second error-free system, called the adaptive responses, can be induced [P. Jeggo, M. Defais, L. Samson, and P. Schendel, Mol. Gen. Genet, 157:1-9, 1977; L. Samson and J. Cairns, Nature (London) 267:281-283, 1977]. The adaptive response is capable of dealing with a much larger amount of alkylation damage than the constitutive system and, when induced, appears to be able to reduce mutagenesis by both decreasing the number of sites available for mutagenesis and delaying the induction of error-prone repair enzymes. Finally, we discuss a model of chemically induced mutagenesis based on these findings which maintains that the observed mutation frequency is dependent on a "race" between these two error-free systems and the two mutagenic pathways.

MOST studies on mutagenesis involve exposing a growing population of cells to a large dose of mutagen for a short period. In a natural environment, however, cells are probably exposed more often to a low concentration of mutagens for long periods. We therefore measured the accumulation of mutants in Escherichia coli cells growing continuously in the presence of very low concentrations of mutagens. Because many mutagens are highly unstable, it was necessary to devise a system in which cells could be continuously exposed to fresh mutagen. To do this we took advantage of the fact that certain bacteria can grow and divide while attached to the underside of a Millipore filter. This system was originally developed by Helmstetter and Cummings1 as a means of producing synchronous cells, because as growth medium is passed through the filter it carries away with it the unattached daughter from each pair of newly divided cells. The mutagen to be studied can therefore be stored in stable conditions and only mixed with the growth medium immediately before being passed through the filter; for example the mutagen N-methyl-N′-nitro-nitro-soguanidine (MNNG) can be stored in citrate buffer at pH 5.0 in which it has a half life of 40 h compared with 2.3 h in synthetic minimal media2,3. We had intended to determine the relationship between ambient concentration of various mutagens and resulting mutation rate. The first mutagen studied was MNNG and the results were totally unexpected.

Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.

The adaptive-response is an inducible repair system of E. coli which reduces the mutagenic and cytotoxic effects of alkylation damage (Samson and Cairns, 1977). In adapted cells (cells exposed to sublethal doses of alkylating agents) the induction of W-reactivation and W-mutagenesis by alkylating agents is almost totally blocked. Despite the fact that adaptation has no detectable effect on UV mutagenesis in E. coli K-12, it does inhibit to some extent the UV and tif-1 mediated induction of SOS functions such as W-reactivation and lambda prophage induction. Furthermore, the kinetics of induction of W-mutagenesis following UV treatment are altered by adaptation. In this case the adaptive-response seems to specifically block the induction of an error-producing W-reactivating capacity which normally would increase soon after UV treatment, while affecting error-free W-reactivating systems to a lesser extent.

N-methyl-N’-nitro-N-nitrosoguanidine, (MNNG), an alkylating agent is a potent mutagen and carcinogen. Like N-methyl-N-nitrosourea, (MNUA) and N-ethyl-N-nitrosourea, (ENUA), but in contrast to most other common alkylating agents, it acts selectively at the replication fork and produces a high proportion of linked mutations (1,2). The mutagenicity of MNNG is probably due to the methylation of DNA bases (possibly at specific sites on bases) (3) but the mechanism by which the premutagenic lesions become converted to stable mutations is not known, and the characteristic action of MNNG at the replication fork is also not understood.

Mutation, cell death and chromosome damage are the ultimate products of the response of mammalian cells to DNA-damaging agents. It is important to determine how cells respond to a continuous exposure to DNA damage (much like we experience in our environment) and to learn about the genetic and molecular events that lie between the incurrence of DNA damage and the final appearance of damaged chromosomes and dead or mutated cells. We now know that bacteria induce specific sets of genes in response to certain types of genetic damage;1 evidence that eukaryotes employ similar strategies is rapidly accumulating. Among other things a detailed understanding of these mechanisms will provide a more sound scientific basis upon which to assess the risk to man from DNA-damaging agents in our environment.

Simple monofunctional alkylating agents such as methylmethane sulphonate (MMS) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) cause a variety of alkylated lesions in DNA, and these lesions can cause the induction of mutation and cell death in both human and bacterial cells. The repair of these DNA alkylation damages reduces cell killing and the induction of mutations and chromosome damage. In E. coli, the repair of 06-methylguanine (06MeG) and 04-methylth5anine (04MeT) specifically prevents these lesions from causing G:C to A:T and A:T to G:C transition mutations, because if left unrepaired these lesions mispair during replication (1–6). On the other hand, the repair of N3-methylpurines (N3MeA and N3MeG) and 02-methylpyrimidines (02MeC and 02MeT) in E. coli specifically prevents cell killing (7–9). The repair of DNA alkylation damage in mammalian cells is somewhat less well understood, and it is not yet clear which alkylated lesions cause mutation and which cause cell death. Like E. coli, mammalian cells can repair 06alkylG, N3alkylA, N3alkylG, 04alkylT, 02alkylT and 02alkylC lesions (10–21). However, because observations have been made with a variety of cell types the results have not always been consistent. For instance, some studies indicated that 04MeT is repaired by rat liver cells (13,20,21), while others did not (22–24). Not all cell lines are able to repair all the alkylated lesions; for instance, CHO and V79 cells (25,26) and some human tumor cell lines (27–29) are unable to repair 06MeG. The ability of these particular cell lines to repair alkylated pyrimidines has not yet been measured. Other human cells, however, have been shown to repair 06MeG (19,27–29), 04alkylT, 02alkylT, 02alkylC (11), N3alkylA, N7alkylG (11,14,15), N3alkylG (15,30) and N7alkylA (15).

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.

Addendum: Standardizing global gene expression analysis between laboratories and across platforms

Background A myriad of new chemicals has been introduced into our environment and exposure to these agents can damage cells and induce cytotoxicity through different mechanisms, including damaging DNA directly. Analysis of global transcriptional and phenotypic responses in the yeast S. cerevisiae provides means to identify pathways of damage recovery upon toxic exposure. Results Here we present a phenotypic screen of S. cerevisiae in liquid culture in a microtiter format. Detailed growth measurements were analyzed to reveal effects on ~5,500 different haploid strains that have either non-essential genes deleted or essential genes modified to generate unstable transcripts. The pattern of yeast mutants that are growth-inhibited (compared to WT cells) reveals the mechanisms ordinarily used to recover after damage. In addition to identifying previously-described DNA repair and cell cycle checkpoint deficient strains, we also identified new functional groups that profoundly affect MMS sensitivity, including RNA processing and telomere maintenance. Conclusions We present here a data-driven method to reveal modes of toxicity of different agents that impair cellular growth. The results from this study complement previous genomic phenotyping studies as we have expanded the data to include essential genes and to provide detailed mutant growth analysis for each individual strain. This eukaryotic testing system could potentially be used to screen compounds for toxicity, to identify mechanisms of toxicity, and to reduce the need for animal testing., Unilever (Firm), National Institutes of Health (U.S.) (grant CA055042), National Institutes of Health (U.S.) (grant ES002109), Swedish Research Council, Spanish Ministry of Science and Innovation