Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

Abstract

Ethylation of poly(dC-dG) poly(dC-dG) with ethyl methanesulfonate (EtMes), a known carcinogen, at increasing molar ratios of EtMes/C-G base pairs progressively stimulated the methyl-accepting ability of the DNA during in vitro methylation by partially purified rat DNA (cytosine-5)-methyltransferase (EC 2.1.1.37). Maximum stimulation was 2-fold over mock-treated DNA when 2.7% of the guanines were modified at the N-7 position, the major site of ethylation by EtMes in DNA. If a CpG site "hemiethylated" at guanine N-7 mimics a hemimethylated CpG site, we calculate that the enzyme has a relative affinity for hemiethylated CpG 18-fold above unmodified CpG. If ethylation of a dioxyphosphate oxygen of the phosphodiester bond is responsible for stimulation, the relative affinity could be much higher, up to 370-fold.DNA methylation at cytosine C-5 in the rare CpG dinucleotide appears to play an important role in regulating the expression of many mammalian genes (reviewed in refs. 1-7) and is being extensively studied as a potential target for chemical and radiation-induced carcinogenesis. A large body of evidence now exists showing that chemical carcinogens and UV irradiation, both of which chemically modify DNA, significantly decrease enzymatic methylation of DNA in vivo and in vitro (8)(9)(10)(11)(12)(13)(14). Demethylation may, therefore, play a role in activation of the cancer cell phenotype as proposed by Holliday (15).By contrast, however, we recently found that one widely used chemical carcinogen, ethyl methanesulfonate (EtMes), inactivated specific gene expression (16) apparently by promoting an increase in enzymatic methylation of cultured cell DNA (17). Methyl methanesulfonate has also been reported to increase the level of 5-methylcytidine in DNA in regenerating rat liver (18). Because EtMes introduces ethyl groups primarily at guanine N-7 and secondarily at the phosphodiester bond (19), it was proposed that EtMes might promote enzymatic methylation (17) by creating a "fraudulent" hemimethylated site by rotation of an ethyl group at guanine N-7 or the phosphodiester in close proximity to cytosine C-5 in CpG sequences in B-DNA. This ethylated site might be recognized as hemimethylated by DNA (cytosine-5)-methyltransferase (MeTase; EC 2.1.1.37) and subsequently undergo methylation. We have tested a prediction of the model that unmethylated CpG sites modified by EtMes will be more efficient substrates for DNA methyltransferase in vitro than unmodified sites, and we report the results here. MATERIALS AND METHODSDNA MeTase. The enzyme was extracted with 0.8 M KCl from purified rat liver and spleen nuclei as described (20,21). The crude nuclear extract in 20 mM NaCi in buffer A (50 mM Tris HCl, pH 7.8/1 mM dithiothreitol) was loaded onto a S-adenosyl-L-homocysteine agarose column (3 ml; Bethesda Research Laboratories); the column was washed with 30 ml of 60 mM KCI/buffer A/1 mM EDTA and 15 ml of the same buffer with 20 ,uM S-adenosylmethionine (AdoMet). Enzyme was eluted with 0.3 M KCI in buffer A/1 mM EDTA a...

Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells.

“…The EMS treatment might then induce aberrant methylation, which shuts off the gene, at a frequency of about 10-3. There is some evidence that DNA-damaging agents induce hypermethylation (9,19); however, they have also been shown in several instances to reduce DNA methylation both in vitro (46) and in vivo (27), and repair tracts may be undermethylated (24). (Note that we cannot exclude the possibility that such hypermethylated variants arise spontaneously at a frequency of 10-3, since only mutagenized isolates were tested in the search for xrs strains).…”

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

“…1 EMS enhances the reversion of HAT' revertants to 6-thioguanine resistance. A major reason for undertaking this study was to test whether EMS could inactivate the activated HPRT+ allele in revertant lines in the same manner it promoted inactivation of the prolactin gene in GH3 cells (9,17,18). Figure 5 shows that a consistent but small increase in the frequency of reversion to 6-thioguanine resistance over the spontaneous frequency was found after EMS treatment HAT+ of either the spontaneous HAT' revertant H23RS-1 (Fig.…”

“…Although the target sequence for the EMS effect has not yet been located, CpG sites ethylated by EMS have about a * Corresponding author. 20-fold increased affinity for the mammalian DNA methyltransferase in vitro (9). A major problem of the GH3 cell system, however, was the absence of genetic selection for prolactin mutants.…”

“…Furthermore, Darlington et al (7) induced a set of mutants in hepatoma cells with MNNG which were remarkably similar to those induced with EMS in GH3 cells although the ability of 5-azacytidine to cause reversion was not tested. In the case of EMS, molecular models have shown that ethyl groups at guanine N7 or one of the free dioxyphosphate oxygens of the phosphodiester bond introduce hydrophobicity into the CpG methylation site in the major groove of B DNA (9) and thus may mimic a hemimethylated site. Furthermore, rat liver DNA methyltransferase had a 20-fold higher affinity for…”

Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine content of normal human bronchial epithelial cells.

“…Farrance and Ivarie (29) also reported increased methyltransferase activity upon ethylation of poly(dC-dG) by ethyl methanesulfonate. However, carcinogen treatment of "immortal" and tumor cells in culture has generally resulted in decreases in genomic m5dC levels (20,29,30 …”