A cytosine . cytosine base paired parallel DNA double helix with thymine . thymine bulges

500 MHz 1H NMR studies using 2D-NOESY indicate that the oligonucleotide d(CTCTCT) at low pH forms a parallel double helix with cytosine . cytosine base pairs and thymine . thymine bulges. This unusual structure may explain the hypersensitivity of S1 nuclease at low pH towards supercoiled plasmids containing d(CT)n inserts.     

Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.

Human cell-free extracts were used to detect activities specifically incising O6-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G:T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell line HT29, containing MGMT, showed incision activities specific for the T strand of m6G:T [and G:T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G:C (or G:C) substrates. Competition experiments showed that the activity was similar to, if not identical with, the activity in human cells that incises G:T mismatches. The incision sites were similar to those recognized by human G:T- or G:A-specific mismatch enzymes, i.e., the phosphodiester bonds both 3' and 5' to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G:T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G:T substrate to a G:T substrate which would serve as a substrate for G:T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G:T (but not the G:T) substrate, that was removed by the addition of synthetic DNA to the reaction.     

Enzymatic methylation of cytosine in DNA is prevented by adjacent O6-methylguanine residues

The effect of O6-alkylation of guanine residues on the enzymic methylation of cytosine has been studied using synthetic oligonucleotides in which all guanines in cytosine-guanine sequences at potentially methylatable sites are replaced by O6-methylguanine. In contrast with the unmodified forms, which showed high acceptance activity for methyl-3H-labeled groups from S-adenosyl-L-[methyl-3H]methionine in the presence of DNA methylase, the modified oligonucleotides were not substrates for the enzyme either in the single-stranded or annealed forms. In view of the importance of cytosine methylation in the down-regulation of certain genes, the potential to affect gene expression by this mechanism may be a contributory factor in the toxic and carcinogenic effects of chemical methylating agents.     

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.     

Reaction of O6-alkylguanine-DNA alkyltransferase with O6-methylguanine analogues: evidence that the oxygen of O6-methylguanine is protonated by the protein to effect methyl transfer.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) repairs the promutagenic O6-methylguanine lesion by transferring the methyl group to a cysteine residue on the protein. A mechanism in which AGT activates the guanyl moiety as a leaving group by protonation of a heteroatom on guanine was probed by reacting AGT with analogues of O6-methylguanine in which the heteroatoms were changed. The initial rates of reaction were measured at various substrate concentrations in 50 mM Hepes, 1 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.8 at 37 degrees C. The kinact (h-1) and Kin (mM) were determined for O6-methylguanine (1.66 +/- 0.19, 1.51 +/- 0.32), 6-methoxypurine (1.07 +/- 0.25, 10.6 +/- 4.2), S6-methyl-6-thioguanine (0.63 +/- 0.04, 1.17 +/- 0.18), 6-methylthiopurine (no reaction), Se6-methyl-6-selenoguanine (1.76 +/- 0.28, 10.6 +/- 5.0), 6-methylselenopurine (2.51 +/- 0.62, 15.7 +/- 6.3), O6-methyl-1-deazaguanine (1.71 +/- 0.34, 14.8 +/- 4.4), O6-methyl-3-deazaguanine (1.90 +/- 0.24, 2.54 +/- 0.59), and O6-methyl-7-deazaguanine (1.97 +/- 0.26, 2.56 +/- 0.72). These results indicate that replacement of the nitrogens does not affect the kinact parameter but the Kin is increased upon removal of the exocyclic amino group and the nitrogen at the 1-position. Replacement of the oxygen with sulfur decreases the kinact, and replacement with selenium increases the Kin. The results are consistent with a mechanism in which O6-methylguanine binds to the active site of AGT with hydrogen bonds to the oxygen, the exocyclic amino group, and the nitrogen at the 1-position of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.     

Methylation of thymine residues during oligonucleotide synthesis.

Thymine residues in an oligodeoxyribonucleotide are subject to methylation at N3 by the internucleotide methyl phosphotriester linkages. This alkylation occurs most rapidly in the presence of a strong base such as DBU, but also takes place, at a much slower rate, during oligonucleotide synthesis.     

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.     

Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs DNA by transferring the methyl group from the 6-position of guanine to a cysteine residue on the protein. We previously found that the Escherichia coli Ada protein makes critical interactions with O6-methylguanine (O6mG) at the N1- and O6-positions. Human AGT has a different specificity than the bacterial protein. We reacted hAGT with double-stranded pentadecadeoxynucleotides containing analogues of O6mG. The second-order rate constants were in the following order (x10(-)5 M-1 s-1): O6mG (1.4), O6-methylhypoxanthine (1.6) > Se6-methyl-6-selenoguanine (0.1) > S6-methyl-6-thioguanine (S6mG) (0.02) > S6-methyl-6-thiohypoxanthine (S6mH), O6-methyl-1-deazaguanine (O6m1DG), O6-methyl-3-deazaguanine (O6m3DG), and O6-methyl-7-deazaguanine (O6m7DG) (all <0.0001). Electrophoretic mobility shift assays were carried out to determine the binding affinity to hAGT. Oligodeoxynucleotides containing O6mG, S6mG and O6m3DG bound to AGT in the presence of competitor DNA with Kd values from 5 to 20 microM, while those containing G, S6mH, O6m1DG, and O6m7DG did not (Kd > 200 microM). These results indicate that the 1-, N2-, and 7- positions of O6mG are critical in binding to hAGT, while the 3- and O6-positions are involved in methyl transfer. These results suggest that the active site of ada AGT is more flexible than hAGT and may be the reason ada AGT reacts with O4mT faster than hAGT.     

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.     

Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli

The qualitative and quantitative features of mutagenesis by two DNA adducts of carcinogenic alkylating agents, O6-methylguanine (m6G) and O4-methylthymine (m4T), were examined in vivo. The deoxyhexanucleotides 5'-GCTAGC-3' and 5'-GCTAGC-3' were synthesized, where the underlined bases are the positions of m4T or m6G, respectively. By use of recombinant DNA techniques, the respective hexanucleotides or an unmodified control were inserted into a six-base gap in the otherwise duplex genome of the Escherichia coli virus M13mp19-NheI. The duplex adducted genome was converted to single-stranded form and introduced into an E. coli strain that was phenotypically normal with regard to m6G/m4T repair, a strain deficient in repair by virtue of an insertion in the gene encoding the Ada-m6G/m4T DNA methyltransferase, or the same two cell lines after challenge with N-methyl-N'-nitro-N-nitrosoguanidine. Treatment with this alkylating agent chemically compromises alkyl-DNA repair functions. The mutation efficiency of m6G was low or undetectable (0-1.7%) in all cell systems tested, owing, we believe, to rapid repair. In striking contrast, the mutagenicity of m4T was high (12%) in cells fully competent to repair alkylation damage and was roughly doubled when those cells were pretreated with N-methyl-N'-nitro-N-nitrosoguanidine to suppress repair. Taken together, these data suggest that m4T is potentially more mutagenic than m6G and, if formed by a DNA methylating agent, may pose a significant threat to the genetic integrity of an organism.     

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.     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Levels of O6-methylguanine acceptor protein in extracts of human breast tumor tissues.

We have measured the abilities of extracts of tissues from human breast tumors to demethylate adducts of O6-meG in exogenous DNA by transfer of the methyl group to an acceptor protein. The results have shown that all 21 specimens examined (including 5 non-neoplastic, 11 malignant tumors and 5 benign growth) contained significant amounts of O6-meG acceptor activity, removing on average 221.1 +/- 2.1 (SEM) fmol O6-meG per mg protein or 10.07 +/- 0.98 (SEM) fmol O6-megG per microgram DNA in the extracts. There were also wide interindividual variations, which were not age-dependent, and there were no significant differences between the non-neoplastic and neoplastic tissues obtained from individuals with benign or with malignant disease. It was estimated that the average number of O6-meG acceptor molecules per cell in normal human breast tissues was calculated as 46,000 +/- 7000 (SEM).     

Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine.

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.     

2-Amino-7-deazaadenine forms stable base pairs with cytosine and thymine.

2-Amino-7-deazaadenine ((AD)A) was incorporated into oligodeoxynucleotides (ODN) and their base-pairing properties with natural nucleobases were investigated. In melting temperature (T(m)) experiments, the duplex containing an (AD)A/C base pair showed a high stability comparable to that containing (AD)A/T base pair. Destabilization of the duplex usually observed for existing degenerate bases was not observed. However, the incorporation efficiency of dCTP was only 1.8% for TTP in single-nucleotide insertion reactions using polymerase.