Apoptotic signaling in response to a single type of DNA lesion, O(6)-methylguanine

Until now, it has been difficult to establish exactly how a specific DNA lesion signals apoptosis because each DNA damaging agent produces a collection of distinct DNA lesions and produces damage in RNA, protein, and lipids. We have developed a system in human cells that focuses on the response to a single type of DNA lesion, namely O(6)-methylguanine (O(6)MeG). We dissect the signaling pathways involved in O(6)MeG-induced apoptosis, a response dependent on the MutSalpha heterodimer that is normally involved in DNA mismatch repair. O(6)MeG triggers robust activation of caspases associated with both death receptor- and mitochondrial-mediated apoptosis. Despite this, O(6)MeG/MutSalpha-triggered apoptosis is only partly dependent on caspase activation; moreover, it is mediated solely by mitochondrial signaling and not at all by death receptor signaling. Finally, while Bcl-2 and Bcl-x(L), negative regulators of mitochondrial-regulated apoptosis, could effectively block O(6)MeG/MutSalpha-dependent apoptosis, they were unable to prevent the cells from ultimately dying.     

O6-methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with O6-methylguanine

Mutations were induced in the ampicillinase gene of a bacteriophage f1/pBR322 chimera both by incorporation of O6-methyl-dGTP opposite T during DNA replication in vitro and by site-directed mutagenesis using O6-methylguanine-containing oligonucleotides. After passage of the DNA through Escherichia coli, analysis of 151 O6-methyl-dGTP-induced mutations indicated a significantly greater number of unmutated mutation sites than expected, whereas the mutated sites generally fit a Poisson distribution. The unmutated sites are assumed to be caused by the inability of some sequences to tolerate the presence of a tetrahedral methyl group within the confines of a Watson-Crick helix (Toorchen, D., and Topal, M.D. (1983) Carcinogenesis 4, 1591-1597). A consensus of the DNA sequences surrounding unmutated mutation sites was derived. The consensus sequence had significant similarity to the region of the rat Harvey ras oncogene containing the N-methyl-N-nitrosourea activated site for transformation (Zarbl, H., Sukumar, S., Arthur, A. V., Dionisio, M.-Z., and Barbacid, M. (1985) Nature 315, 382-385). We propose that direct alkylation at O6 of a guanine present within the consensus sequence may produce a DNA conformation less subject to repair. Mutation by O6-methylguanine-containing oligonucleotides demonstrated that repair of the O6-methylguanine lesions varied at least 3-4-fold with position of the lesion.     

Immunological detection of O6-methylguanine in alkylated DNA

Antibodies to O6-methyldeoxyguanosine were produced in rabbits and utilized in a radioimmunoassay to detect this nucleoside at picomole levels. The specificity of the antibodies was demonstrated by the use of nucleoside analogues as inhibitors in the radioimmunoassay. The antibodies cross-reacted with O6-methylguanosine, O6-methylguanine, and O6-ethylguanosine. There was 10(4) to 10(6) times less sensitivity to inhibition by deoxyadenosine, deoxyguanosine, and guanosine than by O6-methyldeoxyguanosine. The radioimmunoassay also detected O6-methylguanine in DNA alkylated by agents known to produce O6-methylguanine, such as N'-methyl-N-nitrosourea. DNA alkylated with dimethyl sulfate, which does not produce O6-methylguanine in DNA, cross-reacted with the antibodies to a very limited extent. Such an assay system for modified nucleic acid components would be very useful in following the production, persistence, and repair of these lesions in a variety of cells and tissues treated with a broad spectrum of carcinogens and suspected carcinogens.     

Thermodynamic comparison of the base pairs formed by the carcinogenic lesion O6-methylguanine with reference both to Watson-Crick pairs and to mismatched pairs

A set of 10 non-self-complementary nonadeoxyribonucleoside octaphosphates, d(GGTTXTTGG) and d(CCAAYAACC), where X and Y are A, C, G, T, or O6MeG, has been synthesized by a large-scale, automated, phosphoramidite procedure. Purification was effected by reversed-phase HPLC, and the base composition was verified by analytical HPLC after enzymatic degradation to the constituent deoxynucleosides. This set of molecules was designed to allow evaluation of the nearest-neighbor dependence of each base pair. The thermal stability, expressed as Tmax, of each duplex containing one of the O6MeG base pairs, a Watson-Crick pair, or one of the mismatches possible with this set of molecules was determined over a concentration range of 5.7-200 microM. From these data the delta H degree, delta S degree, and delta G degree of each combination were calculated. In general, the relative thermal stabilities observed for the O6-methylguanine combinations confirm our previous findings that the most stable base pair is formed with cytosine rather than thymine and that all O6MeG pairs are much weaker than Watson-Crick base pairs [Kuzmich, S., Marky, L. A., & Jones, R. A. (1983) Nucleic Acids Res. 11, 3393-3404; Gaffney, B. L., Marky, L. A., & Jones, R. A. (1984) Biochemistry 23, 5686-5691]. Moreover, the nine combinations containing O6-methylguanine are all of similar thermal stability, cover a much smaller range in Tmax than do the mismatches, and show little sequence dependence.     

Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT)

The human DNA repair protein O⁶-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O⁶-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O⁶-alkylguanines. One such ODN containing O⁶-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC₅₀ of 0.1 nM.     

Solution structure of a DNA duplex containing a formamide-adenine base pair

The N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside.     

Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines

Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

A common mechanism for repair of O6-methylguanine and O6-ethylguanine in DNA

The mutagenic effects of several ethylating and methylating agents were assessed in Encherichia coli strains that are defective in the adaptive response to alkylating agents. These mutants were either deficient in the response or expressed it constitutively. When expressed, the repair pathway removed the major mutagenic lesion produced by either methylating or ethylating agents. This lesion was almost certainly O⁶-alkylguanine produced by alkylation of DNA, and the mechanism for its removal was characterized in vitro. E. coli cells expressing the adaptive response contain relatively large amounts of a protein that transfers the methyl group from O⁶-methylguanine to one of its own cysteine residues (Olsson & Lindahl, 1980). This methyltransferase was shown to act in an analogous fashion on O⁶-ethylguanine. Incubation of ethylated DNA with purified transferase led to disappearance of the O⁶-ethylguanine residues, and S-ethylcysteine was simultaneously generated in the protein. The greater sensitivity of E. coli wild-type to ethylating than methylating agents may be explained by a slower repair of O⁶-ethylguanine than O⁶-methylguanine and also a weaker ability of ethylating agents to induce the adaptive response.     

Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide lesion

One- and two-dimensional NMR spectroscopy has been used combined with molecular dynamics to determine the fine structure of the DNA duplex 5'-d(AGGAGCCACG).d(CGTGGFTCCT) where F is the N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue which is a ring fragmentation product of thymine. The formamide deoxyribose exists as two isomers with respect to the orientation about the peptide bond. The two isomers (trans and cis) are observed in a ratio 3:2 in solution. For both species, the oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. The formamide residue, whatever the isomer, is intrahelical and can pair with the guanine on the opposite strand with one hydrogen bond. For the cis isomer, the residue adopts a syn orientation and is able to form a second hydrogen bond with the guanine on the 5' side on the same strand. Off-resonance ROESY experiments have been used to investigate the chemical exchange observed at low temperature of the duplex. Conformational exchange has only been found for the oligonucleotide with the formamide residue in the trans conformation.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

Solution structure of duplex DNA containing the mutagenic lesion 1,N(2)-etheno-2'-deoxyguanine

We have used high-resolution NMR spectroscopy and molecular dynamics simulations to determine the solution structure of DNA containing the genotoxic lesion 1, N (2)-etheno-2'-deoxyguanosine (epsilonG), paired to dC. The NMR data suggest the presence of a major, minimally perturbed structure at neutral pH. NOESY spectra indicate the presence of a right-handed helix with all nucleotides in anti, 2'-deoxyribose conformations within the C2'-endo/C1'-exo range and proper Watson-Crick base pair alignments outside the lesion site. The epsilonG residue remains deeply embedded inside the helix and stacks between the flanking base pairs. The lesion partner dC is extrahelical and is located in the minor groove of the duplex, where it is highly exposed to solvent. Upon acidification of the sample, a second conformation at the lesion site of the duplex emerges, with protonation of the lesion partner dC and possible formation of a Hoogsteen base pair. Restrained molecular dynamics simulations of the neutral-pH structure generated a set of three-dimensional models that show epsilonG inside the helix, where the lesion is stabilized by stacking interactions with flanking bases but without participating in hydrogen bonding. The lesion counterbase dC is displaced in the minor groove of the duplex where it can form a hydrogen bond with the sugar O4' atom of a residue 2 bp away.     

Assay for O6-alkylguanine-DNA-alkyltransferase using oligonucleotides containing O6-methylguanine in a BamHI recognition site as substrate.

Double-stranded oligonucleotides, 40 bases in length containing an O6-methylguanine in a BamHI restriction site, were developed as substrates for the determination of human O6-alkylguanine-DNA-alkyltransferase (AGT). The assay proved highly sensitive and quantitative. After incubation of the 5'-end-labeled oligonucleotides with cell homogenates of peripheral blood lymphocytes, the DNA was digested with BamHI. Cleavage with this restriction enzyme did not occur in the O6-methylguanine-containing oligonucleotide unless the fragment was repaired. The cleaved oligonucleotide was separated from the intact parent oligonucleotide by reverse-phase high-performance liquid chromatography. Calculation of the AGT content was achieved by integrating the radioactivity of the peak corresponding to the digested fragment, which is equal to the molar amount of repaired oligonucleotide and corresponds directly to the molar AGT content in the lymphocyte homogenate.     

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.     

O6-methylguanine DNA methyltransferase as a promising target for the treatment of temozolomide-resistant gliomas

Temozolomide (TMZ) is an alkylating agent currently used as first-line therapy for gliomas treatment due to its DNA-damaging effect. However, drug resistance occurs, preventing multi-cycle use of this chemotherapeutic agent. One of the major mechanisms of cancer drug resistance is enhanced activity of a DNA repair enzyme, O⁶-methylguanine-DNA-methyltransferase (MGMT), which counteracts chemotherapy-induced DNA alkylation and is a key component of chemoresistance. MGMT repairs TMZ-induced DNA lesions, O⁶-meG, by transferring the alkyl group from guanine to a cysteine residue. This review provides an overview of recent advances in the field, with particular emphasis on the inhibitors of MGMT and underlying mechanisms. Literature search was performed through PubMed and all relevant articles were reviewed, with particular attention to MGMT, its role in TMZ-resistant gliomas, effects of MGMT inhibitors and the underlying mechanisms. Several strategies are currently being pursued to improve the therapeutic efficacy of TMZ via inhibition of MGMT to reduce chemoresistance and improve overall survival. MGMT may be a promising target for the treatment of TMZ-resistant gliomas.     

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

O⁶-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O⁶-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O⁶-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.