Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

QSAR and Chemical Read-Across Analysis of 370 Potential MGMT Inactivators to Identify the Structural Features Influencing Inactivation Potency.

O6-methylguanine-DNA methyltransferase (MGMT) constitutes an important cellular mechanism for repairing potentially cytotoxic DNA damage induced by guanine O6-alkylating agents and can render cells highly resistant to certain cancer chemotherapeutic drugs. A wide variety of potential MGMT inactivators have been designed and synthesized for the purpose of overcoming MGMT-mediated tumor resistance. We determined the inactivation potency of these compounds against human recombinant MGMT using [3H]-methylated-DNA-based MGMT inactivation assays and calculated the IC50 values. Using the results of 370 compounds, we performed quantitative structure-activity relationship (QSAR) modeling to identify the correlation between the chemical structure and MGMT-inactivating ability. Modeling was based on subdividing the sorted pIC50 values or on chemical structures or was random. A total of nine molecular descriptors were presented in the model equation, in which the mechanistic interpretation indicated that the status of nitrogen atoms, aliphatic primary amino groups, the presence of O-S at topological distance 3, the presence of Al-O-Ar/Ar-O-Ar/R..O..R/R-O-C=X, the ionization potential and hydrogen bond donors are the main factors responsible for inactivation ability. The final model was of high internal robustness, goodness of fit and prediction ability (R2pr = 0.7474, Q2Fn = 0.7375-0.7437, CCCpr = 0.8530). After the best splitting model was decided, we established the full model based on the entire set of compounds using the same descriptor combination. We also used a similarity-based read-across technique to further improve the external predictive ability of the model (R2pr = 0.7528, Q2Fn = 0.7387-0.7449, CCCpr = 0.8560). The prediction quality of 66 true external compounds was checked using the "Prediction Reliability Indicator" tool. In summary, we defined key structural features associated with MGMT inactivation, thus allowing for the design of MGMT inactivators that might improve clinical outcomes in cancer treatment.

The DNA Alkyltransferase Family of DNA Repair Proteins: Common Mechanisms, Diverse Functions.

DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O6-alkylguanine and O4-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O6-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family.

Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of O6-methylguanine and their recognition by MGMT and Atl1.

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.

O6-methylguanine-induced transcriptional mutagenesis reduces p53 tumor-suppressor function.

Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood. In this study, the impact of O6-methylguanine on the transcription fidelity of p53 and the subsequent effects on the protein's function as a regulator of cell death and cell-cycle arrest were examined in human cells. Levels of TM were determined by RNA-sequencing. In cells with active DNA repair, misincorporation of uridine opposite the lesion occurred in 0.14% of the transcripts and increased to 14.7% when repair by alkylguanine-DNA alkyltransferase was compromised. Expression of the dominant-negative p53 R248W mutant due to TM significantly reduced the transactivation of several established p53 target genes that mediate the tumor-suppressor function, including CDKN1A (p21) and BBC3 (PUMA). This resulted in deregulated signaling through the retinoblastoma protein and loss of G1/S cell-cycle checkpoint function. In addition, we observed impaired activation of apoptosis coupled to the reduction of the tumor-suppressor functions of p53. Taking these findings together, this work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for the role of TM in tumorigenesis.

IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide.

Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ → IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage.

MGMT Expression Predicts PARP-Mediated Resistance to Temozolomide.

Melanoma and other solid cancers are frequently resistant to chemotherapies based on DNA alkylating agents such as dacarbazine and temozolomide. As a consequence, clinical responses are generally poor. Such resistance is partly due to the ability of cancer cells to use a variety of DNA repair enzymes to maintain cell viability. Particularly, the expression of MGMT has been linked to temozolomide resistance, but cotargeting MGMT has proven difficult due to dose-limiting toxicities. Here, we show that the MGMT-mediated resistance of cancer cells is profoundly dependent on the DNA repair enzyme PARP. Both in vitro and in vivo, we observe that MGMT-positive cancer cells strongly respond to the combination of temozolomide and PARP inhibitors (PARPi), whereas MGMT-deficient cells do not. In melanoma cells, temozolomide induced an antiproliferative senescent response, which was greatly enhanced by PARPi in MGMT-positive cells. In summary, we provide compelling evidence to suggest that the stratification of patients with cancer upon the MGMT status would enhance the success of combination treatments using temozolomide and PARPi.

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.

Increased N7-methyldeoxyguanosine DNA adducts after occupational exposure to pesticides and influence of genetic polymorphisms of paraoxonase-1 and glutathione S-transferase M1 and T1.

There are concerns about genetic risks associated with long-term exposure to pesticides as these compounds may damage DNA, resulting in mutations that eventually lead to cancer, neurological, and reproductive adverse health effects. This study assessed DNA damage in intensive agricultural workers exposed to pesticides by determining the levels of N7-methyldeoxyguanosine (N7-MedG), an adduct known to be a robust biomarker of recent exposure to chemical methylating agents. A cohort of 39 plastic greenhouse workers was assessed for changes in lymphocyte DNA N7-MedG levels between low level and high level exposures during the course of a spraying season. The contributions of genetic polymorphisms of the pesticide-metabolizing enzymes paraoxonase-1 (PON1) and the glutathione S-transferases, GSTM1 and GSTT1, on N7-MedG levels and other potential confounders were also assessed. N7-MedG increased in the period of high pesticide exposure as compared to the low exposure period (0.23 and 0.18 µmol N7-MedG/mol dG for the unadjusted and adjusted linear mixed models, P = 0.02 and 0.08, respectively). Significant decreased levels of erythrocyte acetylcholinesterase and plasma cholinesterase were observed in the high versus low exposure period in both the unadjusted (2.85 U/g hemoglobin and 213.13 U/L, respectively) and adjusted linear mixed models (2.99 U/g hemoglobin and 230.77 U/L, respectively), indicating pesticide intake. In intensive agriculture workers, higher pesticide exposure increased DNA alkylation levels, further demonstrating the genotoxicity of pesticides in man. In addition, pesticide-exposed individuals with inherited susceptible metabolic genotypes (particularly, null genotype for GSTM1 and the PON1 192R allele) appear to have an increased risk of genotoxic DNA damage. Environ. Mol. Mutagen. 56:437-445, 2015. © 2014 Wiley Periodicals, Inc.

O6-carboxymethylguanine in DNA forms a sequence context-dependent wobble base-pair structure with thymine.

N-Nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue and its occurrence has been linked to diets high in red and processed meats, implying an association with the induction of colorectal cancer. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be mutagenic, inducing G-to-A mutations that may be the molecular basis of increased cancer risk. Previously, the crystal structure of the DNA dodecamer d(CGCG[O(6)-CMG]ATTCGCG) has been reported, in which O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair. In order to further investigate the versatility of O(6)-CMG in base-pair formation, the structure of the DNA dodecamer d(CGC[O(6)-CMG]AATTTGCG) containing O(6)-CMG at a different position has been determined by X-ray crystallography using four crystal forms obtained under conditions containing different solvent ions (Sr(2+), Ba(2+), Mg(2+), K(+) or Na(+)) with and without Hoechst 33258. The most striking finding is that the pairing modes of O(6)-CMG with T are quite different from those previously reported. In the present dodecamer, the T bases are displaced (wobbled) into the major groove to form a hydrogen bond between the thymine N(3) N-H and the carboxyl group of O(6)-CMG. In addition, a water molecule is bridged through two hydrogen bonds between the thymine O(2) atom and the 2-amino group of O(6)-CMG to stabilize the pairing. These interaction modes commonly occur in the four crystal forms, regardless of the differences in crystallization conditions. The previous and the present results show that O(6)-CMG can form a base pair with T in two alternative modes: the Watson-Crick type and a high-wobble type, the nature of which may depend on the DNA-sequence context.

Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells.

BACKGROUND: The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies. Moreover, patients harbouring a wild type BRAF gene (including 25% with NRAS mutations) still require alternative treatment such as chemotherapy. Multiple genetic parameters have been associated with response to chemotherapy, but despite their high frequency in melanoma nothing is known about the impact of BRAF or NRAS mutations on the response to chemotherapeutic agents. METHODS: Using cell proliferation and DNA methylation assays, FACS analysis and quantitative-RT-PCR we have characterised the response of a panel of NRAS and BRAF mutant melanoma cell lines to various chemotherapy drugs, amongst them dacarbazine (DTIC) and temozolomide (TMZ) and DNA synthesis inhibitors. RESULTS: Although both, DTIC and TMZ act as alkylating agents through the same intermediate, NRAS and BRAF mutant cells responded differentially only to DTIC. Further analysis revealed that the growth-inhibitory effects mediated by DTIC were rather due to interference with nucleotide salvaging, and that NRAS mutant melanoma cells exhibit higher activity of the nucleotide synthesis enzymes IMPDH and TK1. Importantly, the enhanced ability of RAS mutant cells to use nucleotide salvaging resulted in resistance to DHFR inhibitors. CONCLUSION: In summary, our data suggest that the genetic background in melanoma cells influences the response to inhibitors blocking de novo DNA synthesis, and that defining the RAS mutation status could be used to stratify patients for the use of antifolate drugs.

Repair and removal of azoxymethane-induced O6-methylguanine in rat colon by O6-methylguanine DNA methyltransferase and apoptosis.

Azoxymethane (AOM) is an alkylating agent that generates mutagenic and carcinogenic O(6)-methylguanine (O(6)meG) adducts in DNA. O(6)meG has been detected in human colonic DNA; hence, understanding the innate cellular events occurring in response to the formation of O(6)meG is important in developing preventive strategies for colorectal cancer. We explored the time-course, dose-response, and kinetics of O(6)meG formation and its removal by the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT), and apoptosis. In rats given AOM (10 mg/kg), the formation of O(6)meG occurs within 2 h of exposure, accompanied by rapid depletion of MGMT activity and followed by the induction of an acute apoptotic response that peaks at 6-8 h. MGMT repair and apoptosis are dependent on AOM dose and O(6)meG load. Apoptosis is initiated only when a high O(6)meG load is present and MGMT activity is fully depleted. AOM, 10 mg/kg, overwhelms MGMT repair for about 96 h and renewed MGMT activity is only observed once O(6)meG is no longer detectable. A threshold for apoptosis is observed at 6 h after 6 mg/kg AOM, when a high O(6)meG persists and MGMT activity is very low. These data suggest that apoptosis is probably triggered by O(6)meG, but only once the capacity of MGMT to repair O(6)meG is exhausted. In the colonic epithelium, apoptosis may be complementary to MGMT, in terms of minimising potentially mutagenic events and maintaining a healthy genome.

Topographical study of O(6)-alkylguanine DNA alkyltransferase repair activity and N7-methylguanine levels in resected lung tissue.

BACKGROUND: Tobacco specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are genotoxic alkylating agents found within cigarette smoke that induce lung adenocarcinomas in animal models. In humans, adenocarcinomas originate most frequently in the lung periphery. The aim of this study was to determine whether peripheral lung has increased susceptibility to the genotoxic effects of alkylating agents by comparing DNA alkylation damage (N7-methylguanine: N7-meG) and repair (O(6)-alkylguanine DNA alkyltransferase: MGMT) in peripheral relative to central lung tissue. METHODS: Macroscopically normal lung tissue, resected from patients undergoing surgery for lung cancer, was sampled at equidistant points from central to peripheral lung along a bronchus. N7-meG levels were determined using an immunoslotblot technique and MGMT activity with a [32P]-labelled oligodeoxynucleotide cleavage assay. RESULTS: A total of 20 subjects were recruited, 12 males and 8 females with a mean age of 68.7±5.8years. There were 14 former and 6 current smokers with a mean smoking exposure of 34.0±18.3packyears. N7-meG (mean 0.75±0.57/10(6)dG, n=65 samples from 14 patients) and MGMT repair (geometric mean 9.57±1.62fmol/µg DNA, n=79 samples from 16 patients) were detected in all samples assayed. MGMT activity increased towards the lung periphery (r=0.28, p=0.023; n=16) with a highly significant association in current (r=0.53, p=0.008; n=6) but not former smokers (r=0.13; p=0.41; n=10). No correlation was seen with N7-meG levels and lung position (r=-0.18; p=0.21; n=14). N7-meG levels were higher in current compared to former smokers reaching significance in two lung positions including peripheral lung (p=0.047). CONCLUSIONS: The findings in this study do not support the hypothesis that peripheral tissue is more susceptible to the genotoxic effects of alkylating agents than central lung tissue. In addition exposure to cigarette smoke reduced the level of MGMT in central bronchial tissue possibly through increased alkylating agent exposure.

Structures of DNA duplexes containing O6-carboxymethylguanine, a lesion associated with gastrointestinal cancer, reveal a mechanism for inducing pyrimidine transition mutations.

N-nitrosation of glycine and its derivatives generates potent alkylating agents that can lead to the formation of O(6)-carboxymethylguanine (O(6)-CMG) in DNA. O(6)-CMG has been identified in DNA derived from human colon tissue, and its occurrence has been linked to diets high in red and processed meats. By analogy to O(6)-methylguanine, O(6)-CMG is expected to be highly mutagenic, inducing G to A mutations during DNA replication that can increase the risk of gastrointestinal and other cancers. Two crystal structures of DNA dodecamers d(CGCG[O(6)-CMG]ATTCGCG) and d(CGC[O(6)-CMG]AATTCGCG) in complex with Hoechst33258 reveal that each can form a self-complementary duplex to retain the B-form conformation. Electron density maps clearly show that O(6)-CMG forms a Watson-Crick-type pair with thymine similar to the canonical A:T pair, and it forms a reversed wobble pair with cytosine. In situ structural modeling suggests that a DNA polymerase can accept the Watson-Crick-type pair of O(6)-CMG with thymine, but might also accept the reversed wobble pair of O(6)-CMG with cytosine. Thus, O(6)-CMG would permit the mis-incorporation of dTTP during DNA replication. Alternatively, the triphosphate that would be formed by carboxymethylation of the nucleotide triphosphate pool d[O(6)-CMG]TP might compete with dATP incorporation opposite thymine in a DNA template.

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase.

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O(6)-CMG is not a substrate for the human version of the DNA damage reversal protein O(6)-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O(6)-alkylguanine lesions by removing alkyl groups from the O(6)-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O(6)-methylguanine (O(6)-MeG) or O(6)-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O(6)-alkyl group and its transfer to the active-site cysteine residue of MGMT. O(6)-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O(6)-CMG is a potential causative agent.

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation-π interactions.

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O(6)-alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O(6)-alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O(6)-alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O(6)-alkylguanine, as determined using molecular mechanics calculations. An unexpected consequence of this feature is the recognition of 2,6-diaminopurine and 2-aminopurine, as confirmed in crystal structures of respective Atl1-DNA complexes. O(6)-Alkylguanine and guanine discrimination is diminished for Atl1 R69A and R69F mutants, and S. pombe R69A and R69F mutants are more sensitive toward alkylating agent toxicity, revealing the key role of Arg69 in identifying O(6)-alkylguanines critical for NER recognition.

Synthesis of oligodeoxyribonucleotides containing a conformationally-locked anti analogue of O6-methyl-2'-deoxyguanosine and their recognition by MGMT and Atl1.

We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system.

Convenient and efficient syntheses of oligodeoxyribonucleotides containing O(6)-(carboxymethyl)guanine and O(6)-(4-oxo-4-(3-pyridyl)butyl)guanine.

O(6)-(carboxymethyl)guanine (O(6)-CMG) and O(6)-(4-oxo-4-(3-pyridyl)butyl)guanine (O(6)-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O(6)-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O(6)-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O(6)-CMG and O(6)-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O(6)-pobG and O (6)-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1.