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cDNA for O6-methylguanine-DNA methyltransferase was isolated by screening rat liver cDNA libraries, using as a probe the human cDNA sequence for methyltransferase. The rat cDNA encodes a protein with 209 amino acid residues. The predicted amino acid sequence of the rat methyltransferase exhibits considerable homology with those of the human, yeast and bacterial enzymes, especially around putative methyl acceptor sites. When the cDNA was placed under control of the lac promoter and expressed in methyltransferase-deficient Escherichia coli (ada-, ogt-) cells, a characteristic methyltransferase protein was produced. The rat DNA methyltransferase thus expressed could complement the biological defects of the E. coli cell caused by lack of its own DNA methyltransferases; e.g. increased sensitivity to alkylating agents in terms of both cell death and mutation induction.

Treatment of cells with a methylating agent, such as methyl methanesulfonate (MMS) and Nmethyl-N-nitrosourea (MNU), yields various methylated bases in DNA (Riazuddin and Lindahl, 1978). Organisms possess mechanisms to repair these methylated bases, which are potentially harmful to their genetic material. O6-methylguanine and O4-methylthymine are repaired by direct removal of their methyl groups by methyltransferases (McCarthy et al., 1984; Teo et al., 1984; Nakabeppu et al., 1985; Potter et al., 1987; Rebeck et al., 1988). Most other lesions, including 3-methyladenine and 3-methylguanine, can be recognized and excised by DNA glycosylases. An apurinic/apyrimidinic (AP) site thus produced (Sagher and Strauss, 1985) is repaired by sequential reactions catalyzed by AP endonuclease, exonuclease, DNA polymerase and ligase. The latter process, called excision repair (base excision repair), functions not only for alkylated bases but also for abnormal bases, such as uracil, in DNA. Since the first report of uracil-DNA glycosylase (Lindahl, 1974), various DNA glycosylases have been found in different organisms (Caradonna et al., 1987; Pierre and Laval, 1986; Crosby et al., 1981; Blalsdell and Warner, 1983; Morgan and Chlebek, 1989; Brent, 1979; Karran and Lindahl, 1980). DNA glycosylases can be defined as enzymes which catalyze hydrolysis of an N-glyco

The Ada protein of Escherichia coli catalyzes transfer of methyl groups from methylated DNA to its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, ada. Using an in vitro reconstituted system, we found that both the sigma factor and the methylated Ada protein are required for transcription of the ada gene. To elucidate molecular mechanisms involved in the regulation of the ada transcription, we investigated interactions of the non-methylated and methylated forms of Ada protein and the RNA polymerase holo enzyme (the core enzyme and sigma factor) with a DNA fragment carrying the ada promoter region. Footprinting analyses revealed that the methylated Ada protein binds to a region from positions −63 to −31, which includes the ada regulatory sequence AAAGCGCA. No firm binding was observed with the non-methylated Ada protein, although some DNase I-hypersensitive sites were produced in the promoter by both types of Ada protein. RNA polymerase did bind to the promoter once the methylated Ada protein had bound to the upstream sequence. To correlate these phenomena with the process in vivo, we used the DNAs derived from promoter-defective mutants. No binding of Ada protein nor of RNA polymerase occurred with a mutant DNA having a C to G substitution at position −47 within the ada regulatory sequence. In the case of a −35 box mutant with a T to A change at position −34, the methylated Ada protein did bind to the ada regulatory sequence, yet there was no RNA polymerase binding. Thus, the binding of the methylated Ada protein to the upstream region apparently facilitates binding of the RNA polymerase to the proper region of the promoter. The Ada protein possesses two known methyl acceptor sites, Cys69 and Cys321. The role of methylation of each cysteine residue was investigated using mutant forms of the Ada protein. The Ada protein with the cysteine residue at position 69 replaced by alanine was incapable of binding to the ada promoter even when the cysteine residue at position 321 of the protein was methylated. When the Ada protein with alanine at position 321 was methylated, it acquired the potential to bind to the ada promoter. These results are compatible with the notion that methylation of the cysteine residue at position 69 causes a conformational change of the Ada protein, thereby facilitating binding of the protein to the upstream regulatory sequence.

Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the −10 and the −35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eight-nucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the −35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the ada-specific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.

Imprinting centers (IC) can be defined as cis-elements that are recognized in the germ line and are epigenetically modified to bring about the full imprinting program in a somatic cell. Two paternally expressed human genes, HYMAI and PLAGL1 (LOT1/ZAC), are located within human chromosome 6q24. Within this region lies a 1-kb CpG island that is differentially methylated in somatic cells, unmethylated in sperm, and methylated in mature oocytes in mice, characteristic features of an IC. Loss of methylation of the homologous region in humans is observed in patients with transient neonatal diabetes mellitus and hypermethylation is associated with a variety of cancers, suggesting that this region regulates the expression of one or more key genes in this region involved in these diseases. We now report that a transgene carrying the human HYMAI/PLAGL1 DMR was methylated in the correct parent-origin-specific manner in mice and this was sufficient to confer imprinted expression from the transgene. Therefore, we propose that this DMR functions as the IC for the HYMAI/PLAGL1 domain.

Using transgenic substrates, we found that the immunoglobulin kappa gene 3' enhancer (E3') acts as a negative regulator in V kappa-J kappa joining. Although the E3' was originally identified as a transcriptional enhancer, it acts in a suppressive manner for recombinational regulation. Base substitution analysis has shown that the PU.1-binding site within the E3' regulates the B/T specificity of V kappa-J kappa joining. In a substrate with a mutated PU.1-binding site (GAGGAA to TCTTCG), V kappa-J kappa joining occurred not only in B cells, but also in T cells. The E3' region is also responsible for determining the pro-B/pre-B specificity of V kappa-J kappa joining. When the E3' region was deleted, kappa gene rearrangement actively occurred at the early pro-B stage of B cell development: nongermline (N) nucleotides were common at recombination junctions.

Reactive oxygen species produced in response to UVR are important in skin tumor development. We have previously reported that deficiency of the Ogg1 gene, encoding the repair enzyme for 8-oxo-7,8-dihydroguanine (8-oxoG), increases skin tumor incidence in mice upon repetitive UVB exposure and modulation of UVB-induced inflammatory response. Spirulina platensis is used as a human food supplement because it contains abundant nutritional and antioxidant components. Therefore, we investigated the inhibitory effects of S. platensis on UVB-induced skin tumor development in Ogg1 knockout-(KO) mice and the wild-type (WT) counterpart. Dietary S. platensis suppressed tumor induction and development in both genotypes compared with our previous data without S. platensis . Induction of erythema and ear swelling, one of the hallmarks of UVB-induced inflammatory responses, was suppressed in the skin of Ogg1 -KO mice and albino hairless mice fed with dietary S. platensis. Compared with untreated mice, S. platensis -administered mice showed significantly reduced 8-oxoG formation in the skin after UVB exposure. Moreover, we found that S. platensis effectively downregulated the signal proteins p38 mitogen-activated protein kinase, stress-activated protein kinase/c-Jun N-terminal kinase, and extracellular signal–regulated kinase after UVB exposure especially in Ogg1 -KO mice. Our results suggest that S. platensis exerts antitumor effects against UVB irradiation in the skin through its anti-inflammatory and antioxidant effects.

The adaptive response is a cellular process to induce DNA repair enzymes in response to a challenge of alkylating agents. In this process Ada protein, the product of the ada gene, plays a major role; it accepts the methyl groups of the methylated DNA at the cysteine residues of its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, thereby triggering induction of the whole process. In addition to this DNA-mediated activation of Ada protein, we have proposed an alternative mechanism which involves direct methylation of Ada protein by methylating agents (Takahashi, K., and Kawazoe, Y. (1987) Biochem. Biophys. Res. Commun. 144, 447-453). Here we present evidence that Ada protein is activated as a transcriptional regulator through a direct methylation by certain methylating agents. A purified preparation of Ada protein was treated with various methylating agents, and the activity to promote transcription of the ada gene was determined using an in vitro reconstituted system. The ada-specific RNA was produced when the Ada protein treated with methyl methanesulfonate or with methyl iodide was present in the reaction mixture. The Ada protein treated with N-methyl-N-nitrosourea did not show such an activity. It is, therefore, suggested that the adaptive response induced by chemoselective methylating agents such as methyl iodide might be due, at least in part, to the direct methylation of the constitutive Ada protein present in the cell.

We constructed a recombinant plasmid carrying a gene that suppresses tag mutation. To overproduce its gene product, a 0.8-kilobase DNA fragment which carries the gene was placed under the control of the lac promoter in pUC8. 3-Methyladenine-DNA glycosylase activity in cells carrying such plasmids (pCY5) was 450-fold higher than that of wild type strain, on exposure to isopropyl-beta-D-thiogalactopyranoside. From an extract of such cells, the enzyme was purified to apparent physical homogeneity, and the amino acid composition and the amino-terminal amino acid sequence of the enzyme were determined. The data were in accord with nucleotide sequence of the gene, determined by the dideoxy method. It was deduced that 3-methyladenine-DNA glycosylase I comprises 187 amino acids and its molecular weight is 21,100, consistent with the value estimated from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. Only 3-methyladenine was excised from methylated DNA by the purified glycosylase. These results show that the tag is the structural gene for 3-methyladenine-DNA glycosylase I.

Alkylating agents are potent mutagens and carcinogens, and organisms respond in a complex manner to these agents. Growth of Escherichia coli in the presence of low levels of simple alkylating agents, such as N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), results in a marked increase in resistance of cells to both the mutagenic and the lethal effects of challenging doses of the same agents (24). This adaptive response is distinct from previously characterized pathways of DNA repair, particularly from the SOS response, another inducible effect resulting from DNA damage. The adaptation does not lead to expression of the SOS functions, and recA and lexA mutant cells that are unable to perform SOS repair can be adapted to MNNG (8, 27).