Construction and characterization of extrachromosomal probes for mutagenesis by carcinogens: site-specific incorporation of O6-methylguanine into viral and plasmid genomes.

Organic synthesis and recombinant DNA technology were used to situate a putatively premutagenic DNA lesion, O6-methylguanine (O6MeGua), at a specific location in the genomes of two bacterial viruses, M13mp8 and phi X174, and of the bacterial plasmid pBR322. In each genome the first guanine residue in the unique recognition sequence for restriction endonuclease Pst I (5'-C-T-G-C-A-G-3') was replaced with O6MeGua. This was accomplished by ligating a chemically synthesized tetranucleotide, 5'-pTpm6GpCpA-3', into a circular, genome-length heteroduplex in which the four internal nucleotides of the Pst I recognition site had been removed from one strand of the DNA double helix (ligation yield, approximately equal to 50%). It was established that the tetranucleotide was located specifically at the Pst I site and that the presence of O6MeGua rendered the ligation product resistant to cleavage by Pst I. Sensitivity of the genome to Pst I was restored upon treatment with purified Escherichia coli O6MeGua DNA-methyltransferase, a repair protein that removes the methyl group from DNA-bound O6MeGua. This result, in combination with other data, showed unambiguously that O6MeGua was incorporated with high yield into the Pst I recognition sequence.     

Removal of O6-methylguanine from DNA by human liver fractions.

In in vitro assays using methylated DNAs as substrates, human liver fractions were shown to be able to catalyze the removal of O6-methylguanine. The amount of removal was proportional to the amount of protein added, and the loss of O6-methylguanine occurred with stoichiometric formation of guanine in the DNA and S-methylcysteine in protein. This indicates that human liver contains a protein similar to that previously found in bacteria exposed to alkylating agents. This protein acts as a transmethylase, transferring the intact methyl group from O6-methylguanine in DNA to a cysteine residue on that protein. A similar activity is present in rodent liver, but it was found that human liver was about 10 times more active in carrying out this reaction. In contrast, there was no difference between the human and rat liver extracts in catalyzing the loss of another methylation product, 7-methylguanine, from alkylated DNA. The liver is the organ most likely to be alkylated after exposure to exogenous potential alkylating agents such as dimethylnitrosamine. The present results show that human liver has a significant capacity to repair O6-methylguanine in DNA, which has been implicated as a critical product in carcinogenesis and mutagenesis.     

Repair of O6-methylguanine in adapted Escherichia coli.

Cells exposed to sublethal concentrations of simple alkylating agents develop resistance to their mutagenic effects. This results from the induction of a system that we have called the adaptive response. During exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Escherichia coli cells induced for the adaptive response accumulate substantially less O6-methylguanine in their DNA than control cells. If O6-methylguanine does form, adapted cells possess a repair system for removing it from their DNA. The capacity of this system is limited and the system ceases to function when too much alkylation has occurred. From this point onwards O6-methylguanine starts to accumulate, and the cells begin to develop mutations at a rate directly proportional to their rate of O6-methylguanine accumulation. Our data support the idea that the O6 methylation of guanine accounts for most MNNG-induced mutagenesis.     

The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.

A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.     

Repair of DNA lesion O 6-methylguanine in hepatocellular carcinogenesis

Evidence from both experimental carcinogenesis and studies in human cirrhotic liver suggest that defective repair of the promutagenic DNA base lesion,O⁶-methylguanine, is a factor in the multistep process of hepatocellular carcinogenesis. Ubiquitous environmental alkylating agents such as N-nitroso compounds can produce O⁶-methylguanine in cellular DNA. Unrepaired,O⁶-methylguanine can lead to the formation of G → A transition mutations, a known mechanism of human oncogene activation and tumour suppressor gene inactivation. Combined treatment of rodents with an agent producing O⁶-methylguanine in DNA, and an agent promoting cell proliferation, leads to development of hepatic nodules and hepatocellular carcinoma (HCC), cell division, hence DNA replication, being required for the propagation of tumorigenic mutation(s) in hepatocyte DNA. The paramount importance of O⁶-methylguanine in hepatocellular carcinogenesis is indicated by the observation that transgenic mice engineered to have increased hepatic levels of repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) are significantly less prone to hepatocellular carcinogenesis following alkylating agent treatment. Cirrhosis is a universal risk factor for development of human HCC, and a condition that is characterized by increased hepatocyte proliferation as a result of tissue regeneration. Levels of the human repairing enzyme for O⁶-methylguanine were found to be significantly lower in cirrhotic liver than in normal tissue. In accord with findings from animal models, this suggested a mechanism in which persistence of O⁶-methylguanine due to defective DNA repair by MGMT, together with increased hepatocyte proliferation, might lead to specific gene mutation(s) and hepatocellular carcinogenesis. Screening for the presence and persistence of O⁶-methylguanine in human DNA presently involves formidable technical difficulty. Indications are that such limitations might be overcome by the use of an ultrasensitive method such as immuno-polymerase chain reaction (PCR). This approach should allow parallel measurement of DNA adduct and repair enzyme in routine liver biopsy samples. It might also enable investigation of O⁶-methylguanine in human genes specifically associated with hepatocellular carcinogenesis. Given the wide variation in human MGMT levels observed between individuals, tissues, and cells, this technology should be adapted to permit the ultrasensitive localisation and measurement of adducts and repairing enzyme in liver biopsy tissue sections. Ability to ultrasensitively measure O⁶-methylguanine, and its repair enzyme, should prove valuable in the risk assessment of cirrhotic patients for developing hepatocellular carcinoma.     

Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.

An in vitro system in which bacteriophage T7 DNA is replicated and efficiently packaged into procapsids to form viable phage has been used to examine mutagenesis. The fidelity of replication was assayed both by measuring reversion of an amber mutation in an essential gene and by generation of temperature-sensitive mutants among the phage produced in vitro. Under standard reaction conditions, the fidelity of DNA replication is about equal to that normally found in vivo. However, when O6-methyldeoxyguanosine triphosphate is included in the reaction, O6-methylguanine is incorporated into newly synthesized DNA and the mutation frequencies increase 10- to 70-fold over the control. These experiments demonstrate in vitro mutagenesis with the T7 DNA replication-packaging system and provide more direct evidence for the premutagenic role of O6-methylguanine.     

Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.

The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m6G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment. These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.     

Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine.

The base-pairing interactions of promutagenic O6-methylguanine (O6-MeGua) with cytosine and thymine in deuterated chloroform were investigated by 1H NMR spectroscopy. Nucleosides were derivatized at hydroxyl positions with triisopropylsilyl groups to obtain solubility in nonaqueous solvents and to prevent the ribose hydroxyls from forming hydrogen bonds. We were able to observe hydrogen-bonding interactions between nucleic acid bases in a solvent of low dielectric constant, a condition that approximates the hydrophobic interior of the DNA helix. O6-MeGua was observed to form a hydrogen-bonded mispair with thymine. Whereas O6-MeGua did not form hydrogen bonds with cytosine (via usual, wobble, or unusual tautomeric structures), it did form a 1:1 hydrogen-bonded complex with protonated cytosine. The pairing of unprotonated cytosine in chloroform is thus consistent with the known preference of O6-MeGua for thymine over cytosine in polymerase reactions. In contrast, the pairing of protonated cytosine is consistent with the greater stability of oligonucleotide duplexes containing cytosine.O6-MeGua as compared with thymine.O6-MeGua base pairs [Gaffney, B. L., Markey, L. A. & Jones, R. A. (1984) Biochemistry 23, 5686-5691]. Our observation that cytosine must be protonated in order to pair with O6-MeGua suggests that the cytosine.O6-MeGua base pair in DNA is stabilized by protonation of cytosine. Through this mechanism, methylation at the O6 position of guanine in double-stranded DNA could promote cross-strand deamination of cytosine (or 5-methylcytosine) to produce uracil (or thymine).     

Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.

Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.     

O(6)-methylguanine DNA methyltransferase activity in diabetic patients

In the present study, we evaluated O(6)-methylguanine-DNA methyltransferase (MGMT) activity in diabetic patients. The study was performed on 27 patients with Type 1 diabetes, and 42 with Type 2 diabetes. Patients with complications were excluded from the study. 36 non-diabetic volunteers, non-smokers who do not consume alcoholic beverage, were chosen from the medical staff as control subjects. MGMT activity was measured by the transfer of radiolabeled methyl groups from a prepared methylguanine-DNA substrate to the enzyme fraction of leukocyte extract. Leukocyte MGMT activity was significantly reduced in both Type 1 and Type 2 diabetes patients as compared with control subjects (P<0.001). The present study demonstrates decreased MGMT activity in leukocytes from patients with Type 1 and Type 2 diabetes.     

In vivo aminoacylation of human and Xenopus suppressor tRNAs constructed by site-specific mutagenesis.

Amber suppressor tRNA genes were constructed by site-specific mutagenesis of the anticodons of human lysine-inserting tRNA (tRNA(Lys)) and glutamine-inserting tRNA (tRNA(Gln)) genes, and a Xenopus laevis tyrosine-inserting tRNA (tRNA(Tyr)) gene. As previous in vitro studies in prokaryotes have shown that substitution of nucleotides in the anticodon region can profoundly affect tRNA aminoacylation, it is important to determine whether the mutation affects aminoacylation of these eukaryotic tRNAs. We present a method for quantitating the tRNA aminoacylation in vivo in mammalian cells, and we have determined that the suppressor tRNA(Tyr) is fully aminoacylated and suppressor tRNA(Lys) and tRNA(Gln) are aminoacylated 40-50% and 80%, respectively. This in vivo method of estimating aminoacylation may be applied to other mutations in the tRNA genes.     

uvrA and recA mutations inhibit a site-specific transition produced by a single O6-methylguanine in gene G of bacteriophage phi X174.

Using site-specific mutagenesis, we have examined the mutagenic activity in vivo of O6-methylguanine or O6-n-butylguanine located at a preselected site in gene G of bacteriophage phi X174. The experiments were designed so that the phage mutant produced by a targeted transition from either of these alkylated derivatives would be recognizable by a simple plaque assay. Spheroplasts derived from normal and repair-deficient cells were transfected, and the lysates were screened for mutant virus. In cells with normal repair, DNA carrying the methylguanine produced the expected transition in 15% of the total phage; DNA carrying the butylguanine produced the same mutation in 0.3% of the phage. In cells deficient in excision repair (uvrA) the transition frequency went up by a factor of 8 for O6-butylguanine and down by a factor of 40 for O6-methylguanine. In cells deficient in recombination (recA), the transition frequency increased 1.5-fold for butylguanine and decreased by a factor of 8 for methylguanine. The data show that both methyl- and butylguanine produce site-directed transitions in phi X174; the transition occurs in recA cells; the frequency of the transition is influenced by both recA and uvrA mutations; the recA and uvrA mutations alter the transition frequency for methylguanine and butylguanine in opposite directions.     

Protection of hematopoietic cells against combined O6-benzylguanine and chloroethylnitrosourea treatment by mutant forms of O6-methylguanine DNA methyltransferase

This report demonstrates that expression of the P140A O6-methylguanine DNA methyl transferase (MGMT) mutant via retrovirus-mediated gene transfer leads to significant, but modest, resistance of cells to both 6-benzylguanine (6-BG) depletion and treatment with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). Expression of the P140A/G156A double mutant appeared to be associated with reduced or unstable protein in hematopoietic cells.     

Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage 29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the 29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.     

O6-methylguanine repair in liver cells in vivo: Comparison between G1- and S-phase of the cell cycle

Summary To compare the formation and persistance of alkylated DNA bases in the G₁- and S-phase compartments in liver in vivo, regnerating rat liver was exposed to [¹⁴C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl ¹⁴C]-N-nitrosourea (3.3 mg/kg, intraportal injection) during the G₁ phase of the cell cyle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl-N-nitrosourea the molar fractions of 7-methylguanine (7megua), O⁶-methylguanine (O⁶megua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the O⁶megua/7megua ratio revealed a loss of O⁶megua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of O⁶megua in S-phase cells was confirmed after intraportal administration of N-methyl-N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the O⁶megua/7megua ratio was constant in G₁ cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher O⁶megua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of O⁶megua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites.Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdaySupported by grants from the Deutsche Forschungsgemeinschaft and the Dr. Mildred Scheel-Stiftung     

Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis.

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.     

Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase.

A plasmid was constructed in which a single guanine residue was replaced with either O6-methylguanine or O6-ethylguanine, two of the DNA adducts formed by carcinogenic alkylating agents. The vectors were introduced in parallel into a pair of Chinese hamster ovary cells, in which one member of the pair was deficient in the repair enzyme O6-alkylguanine-DNA alkyltransferase (mex-) and the other was proficient in this activity (mex+). The vectors integrated into and replicated within the respective host genomes. After intrachromosomal replication, the DNA sequence in the vicinity of the originally adducted site of each integrated vector was amplified from the host genome by using the polymerase chain reaction and was analyzed for mutations. High levels of mutation were observed from the O6-methylguanine- and O6-ethylguanine-containing vectors replicated in mex- cells (approximately 19% for O6-methylguanine and approximately 11% for O6-ethylguanine). DNA sequencing revealed the induced mutations to be almost exclusively G----A transitions. By contrast, little or no mutagenesis was detected when the adducted vectors were introduced into mex+ cells, indicating the significant role of the O6-alkylguanine-DNA alkyltransferase in the repair of O6-methylguanine and O6-ethylguanine in these mammalian cells.     

Persistence of viral genome into late stages of murine myocarditis detected by polymerase chain reaction.

BACKGROUND. Enteroviruses have been considered as the most common etiologic agents in clinical myocarditis and dilated cardiomyopathy; however, their pathogenetic role remains unknown. Hence, the relation of viral replication and development of cardiomyopathy has been determined in a murine model of myocarditis by evaluating the persistence of viral genome during acute and chronic stages of myocarditis by means of Northern blot hybridization and polymerase chain reaction (PCR). METHODS AND RESULTS. DBA/2 mice (n = 146) were injected peritoneally with 10 plaque-forming units of encephalomyocarditis (EMC) virus, and the control mice (n = 33) were injected with normal saline. Animals were randomly killed at 4, 7, 10, 14, 21, 28, 35, and 42 days after infection. Histology revealed acute myocardial necrosis with massive inflammatory cell infiltrate peaking on day 14 followed by increasing fibrosis and declining chronic inflammation features compatible with dilated cardiomyopathy between days 21 and 42. Northern blot analysis of control and infected hearts showed detectable viral RNA in the infected hearts initially at day 4, peaking by day 7, diminishing between day 7 and day 14, and absent at day 21 and day 28. However, potential viral remnants present in low quantities and undetectable by Northern blot were further detected by PCR followed by confirmation with an internal oligonucleotide probe after day 14 up to day 42. CONCLUSIONS. Viral RNA signals on Northern blot showed a strong correlation with massive myocyte necrosis on day 14, but the viral RNA fragment was consistently detectable into late stages of cardiomyopathy on days 21, 28, 35, and 42 by PCR. This indicated that the mature virions are fully developed early in infection and are capable of persisting in the myocardium after virus-mediated myocytolysis stage. Therefore, PCR is an extremely sensitive method for detecting residual viral genome and viral persistence in the myocardium and may offer insights into the pathogenesis of chronic myocarditis leading to dilated cardiomyopathy.