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.     

Effect of sequence context on O(6)-methylguanine repair and replication in vivo.

Understanding the origins of mutational hotspots is complicated by the intertwining of several variables. The selective formation, repair, and replication of a DNA lesion, such as O(6)-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment. A nearest-neighbor analysis was used to address the contribution of sequence context on m(6)G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo. Sixteen M13 viral genomes with m(6)G flanked by all permutations of G, A, T, and C were constructed and individually transformed into repair-deficient and repair-proficient isogenic cell strains. The 16 genomes were introduced in duplicate into 5 different cellular backgrounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay. The Ada methyltransferase demonstrated strong 5' and 3' sequence-specific repair of m(6)G in vivo. The Ada 5' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6)G, N = any base), while the Ada 3' preference decreased in the order: NX(T/C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90%. The Ogt methyltransferase provided MFs ranging from 10% to 25%. As was demonstrated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN context. When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA polymerase places T opposite m(6)G during replication irrespective of the local sequence environment.     

Repair of O6-ethylguanine DNA lesions in isolated cell nuclei. Presence of the activity in the chromatin proteins

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6-ethylguanine and 7-ethylguanine during a subsequent incubation at 37 degrees C. The rate of O6-ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6-ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7-ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7-ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase. The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6-ethylguanine from an added ethylated DNA. No free O6-ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6-ethylguanine were released either, indicating that the disappearance of O6-ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease. Activities capable of removing O6-ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2-3-times the repair activity of the chromatin proteins.     

Inactive O6-methylguanine-DNA methyltransferase in human cells.

A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage lambda N protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.     

Mutant Escherichia coli Ada proteins simultaneously defective in the repair of O6-methylguanine and in gene activation.

The activated Ada protein triggers expression of DNA repair genes in Escherichia coli in response to alkylation damage. Ada also possesses two distinct suicide alkyltransferase activities, for O6-alkylguanines and for alkyl phosphotriesters in DNA. The mutant Ada3 and Ada5 transferases repair O6-methylguanine in DNA 20 and 3000 times more slowly, respectively, than the wild-type Ada protein, but both exhibit normal DNA phosphotriester repair. These same proteins also exhibit delayed and sluggish induction of the ada and alkA genes. Since the C-terminal O6-methylguanine methyltransferase domain of Ada is not implicated in the direct binding of specific DNA sequences, this part of the Ada protein is likely to play an alternative mechanistic role in gene activation, either by promoting Ada dimerization, or via direct contacts with RNA polymerase.     

Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.     

人MGMT基因Cdna的克隆表达及表达蛋白修复功能的鉴定

克隆了Hela细胞O6 甲基鸟嘌呤 DNA 甲基转移酶 (MGMT)基因的cDNA序列 ,该序列与国外发表的cDNA完全一致。将此cDNA插入原核表达载体pET 2 1a后转化大肠杆菌BL2 1(DE3)获得表达的重组菌株pET 2 1a MGMT E .coliBL2 1(DE3) ,经IPTG诱导后产生分子量为 2 4kD的蛋白质。烷化类诱变剂致死突变实验表明 ,MGMT蛋白的表达能修复受体菌DNA分子因烷化类诱变剂导致的突变。     

Alternative pathways for the in vivo repair of O6-alkylguanine and O4-alkylthymine in Escherichia coli: the adaptive response and nucleotide excision repair.

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.     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

Mismatch repair proteins collaborate with methyltransferases in the repair of O(6)-methylguanine

DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O(6)-methylguanine (O(6)mG), which stably pairs with thymine during replication and thereby creates a promutagenic O(6)mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O(6)mG:T mismatches can lead to cell death or result in G:C-->A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O(6)mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O(6)mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O(6)mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O(6)mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O(6)mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O(6)mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.     

Redesigning the substrate specificity of human O(6)-alkylguanine-DNA alkyltransferase. Mutants with enhanced repair of O(4)-methylthymine.

Human O(6)-alkylguanine-DNA alkyltransferase (MGMT) repairs potentially cytotoxic and mutagenic alkylation damage at the O(6)-position of guanine and the O(4)-position of thymine in DNA. We have used random sequence mutagenesis and functional complementation to obtain human MGMT mutants that are resistant to the MGMT inhibitor, O(6)-benzylguanine [Encell, L. P., Coates, M. M., and Loeb, L. A. (1998) Cancer Res. 58, 1013-1020]. Here we describe screening of O(6)-benzylguanine-resistant mutants for altered substrate specificity, i.e., for an increased level of utilization of O(4)-methylthymine (m(4)T) relative to that of O(6)-methylguanine (m(6)G). One mutant identified by the screen, 56-8, containing eight substitutions near the active site (C150Y, S152R, A154S, V155G, N157T, V164M, E166Q, and A170T), was purified and characterized kinetically. The second-order rate constant for repair of m(4)T by the mutant was up to 11.5-fold greater than that of WT MGMT, and the relative m(4)T specificity, k(m(4)T)/k(m(6)G), was as much as 75-fold greater. In competition experiments with both substrates present, the mutant was 277-fold more sensitive to inhibition by m(4)T than WT MGMT. This mutant, and others like it, could help elucidate the complex relationship between adduction at specific sites in DNA and the cytotoxicity and mutagenicity of alkylating agents.     

O6-methylguanine-DNA methyltransferase deficiency in developing brain: implications for brain tumorigenesis

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O(6)-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT(-) phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P相似文献   

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

Coffee and its chemopreventive components Kahweol and Cafestol increase the activity of O6-methylguanine-DNA methyltransferase in rat liver--comparison with phase II xenobiotic metabolism

A lower rate of colon cancer was observed in consumers of coffee with a high content of the diterpenes Kahweol and Cafestol (K/C). In animal models, K/C have been found to protect against the mutagenic/carcinogenic effects of compounds such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), aflatoxin B1, and 7,12-dimethylbenz[a]anthracene. Thus far, such chemoprotection by K/C has been attributed to modifications of xenobiotic metabolism, e.g. enhanced detoxification by UDP-glucuronosyltransferase (UDPGT) and/or glutathione transferase (GST). In the present study, we investigated the potential of several coffee-related treatments (K/C [1:1], Cafestol-alone, Turkish coffee) to modify the expression level of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) which is involved in the reversal of the precarcinogenic DNA damage O(6)-alkylguanine induced by alkylating agents. The results show that, in the male F344 rat, K/C and Cafestol increase hepatic MGMT in a dose-dependent manner up to a maximum of 2.6-fold at 0.122% K/C in the feed. Turkish coffee led to enhancements of up to 16%, the more moderate increase being associated with the lower estimated K/C intake through the beverage. In the livers of the rats receiving Turkish coffee, we also found 10-30% increases in several GST-related parameters (overall GST, GST-pi, glutathione, gamma-glutamylcysteine-synthetase) and a two-fold increase in UDPGT activity. Dose-response studies with K/C revealed that MGMT increased in parallel with three of the four GST-related parameters whereas the dose-response curves of UDPGT and of GST-pi activity displayed a steeper slope. Increased expression level of MGMT may extend the antimutagenic/anticarcinogenic potential of coffee components to protection against DNA alkylating agents.     

Stress factors affecting expression of O6-methylguanine-DNA methyltransferase mRNA in rat hepatoma cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is decisively involved in protecting mammalian cells against genotoxic effects of alkylating carcinogens. We analysed regulation of MGMT expression after exposing rat hepatoma H4IIE cells to various 'stress' factors. Treatments that damage DNA such as alkylation, hydrogen peroxide, ultraviolet or X-ray exposure, as well as restriction enzymes introduced into cells by electroporation or arrest of replication by hydroxyurea significantly induced MGMT mRNA (2.5 to 5-fold). Slight induction (up to 2.5-fold) was observed after heat shock or cadmium/zinc treatment. No or only a very weak induction (less than 1.5-fold) was observed after treatment with 6-thioguanine, 5-azacytidine, transfection of methylated DNA, depletion of MGMT by feeding with O6-methylguanine or O6-benzylguanine, serum starvation and feeding of starved cells, cAMP, TPA and dexamethasone treatment. Inhibitors of protein kinases, H8 and H9, induced MGMT mRNA. On the other hand, an inhibitor of phosphatases (sodium vanadate) prevented induction of MGMT by N-methyl-N'-nitro-N-nitrosoguanidine. The data indicate that DNA breaks are an ultimate signal for MGMT mRNA induction and that protein phosphorylation is involved in regulating MGMT expression.     

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.     

The synthesis and properties of tricyclic analogues of S6-methylthioguanine and O6-methylguanine

The syntheses of novel tricyclic pyrrolo[2,3-d]pyrimidine analogues of O(6)-methylguanine and S(6)-methylthioguanine are described. The crystal structures and pK(a) values of these analogues are reported. In a standard substrate assay with the human repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) only the oxygen-containing analogue displayed activity.