LINE1 methylation levels in pre-diagnostic leukocyte DNA and future renal cell carcinoma risk

Higher levels of LINE1 methylation in blood DNA have been associated with increased kidney cancer risk using post-diagnostically collected samples; however, this association has never been examined using pre-diagnostic samples. We examined the association between LINE1 %5mC and renal cell carcinoma (RCC) risk using pre-diagnostic blood DNA from the United States-based, Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) (215 cases/436 controls), and the Alpha-tocopherol, Beta-carotene Cancer Prevention Study (ATBC) of Finnish male smokers (191 cases/575 controls). Logistic regression adjusted for age at blood draw, study center, pack-years of smoking, body mass index, hypertension, dietary alcohol intake, family history of cancer, and sex was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) using cohort and sex-specific methylation categories. In PLCO, higher, although non-significant, RCC risk was observed for participants at or above median methylation level (M2) compared to those below the median (M1) (OR: 1.37, 95% CI: 0.96–1.95). The association was stronger in males (M2 vs. M1, OR: 1.54, 95% CI: 1.00–2.39) and statistically significant among male smokers (M2 vs. M1, OR: 2.60, 95% CI: 1.46–4.63). A significant interaction for smoking was also detected (P-interaction: 0.01). No association was found among females or female smokers. Findings for male smokers were replicated in ATBC (M2 vs. M1, OR: 1.31, 95% CI: 1.07–1.60). In a pooled analysis of PLCO and ATBC male smokers (281cases/755controls), the OR among subjects at or above median methylation level (M2) compared to those below the median (M1) was 1.89 (95% CI: 1.34–2.67, P-value: 3 x 10^–4); a trend was also observed by methylation quartile (P-trend: 0.002). These findings suggest that higher LINE1 methylation levels measured prior to cancer diagnosis may be a biomarker of future RCC risk among male smokers.     

Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase

The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosyl-methionine to the M.EcoKI:DNA complex, the 2-aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.     

M33 condenses chromatin through nuclear body formation and methylation of both histone H3 lysine 9 and lysine 27

Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases.     

Tissue culture‐induced genomic alteration in maize (Zea mays) inbred lines and F1 hybrids

Genomic alteration is a common phenomenon associated with plant tissue culture, which often encompasses genetic changes and epigenetic modifications (e.g. cytosine methylation). Here, we studied genomic alteration in maize by assessing calli and regenerated plants derived from three inbred lines (M17, J7 and JC) and two pairs of reciprocal F1 hybrids (pair I: M17/J7 and J7/M17 and pair II: M17/JC and JC/M17). By employing two molecular markers, the amplified fragment length polymorphism and methylation‐sensitive amplified polymorphism, we found that both types of genomic alterations occurred in calli and regenerated plants of all the studied maize inbred lines and F1 hybrids, but the extent and pattern of changes varied substantially across the genotypes. Among the three inbred lines, M17 showed markedly higher frequencies of both genetic (from 2.1% to 3.8%) and methylation alterations (from 6.5% to 9.9%, by adding up the various patterns) than the other two lines which showed similar frequencies for both types of alterations (genetic: 0.5–1.8%, methylation: 2.1–3.7%). Of the two F1 hybrid pairs, while pair I showed genetic variation frequencies similar to that of the inbred parent with lower changing frequency and pair II was intermediate of those of the parents, both pairs showed frequencies of methylation alteration more or less intermediate of those of their inbred parental lines. Parent‐of‐origin effects in both genetic and methylation changes were detected in only one of the hybrid pairs (primarily pair II) for a given changing pattern. Statistical testing confirmed the genotypic difference in both genetic and methylation (hypomethylation) alterations among the regenerants. Taken together, it could be concluded that the frequency and pattern of both genetic and cytosine methylation alterations in maize tissue culture were largely genetic context‐dependent traits, but stochasticity also played an important part. F1 hybrids were not significantly more stable than their inbred parental lines under tissue culture conditions.     

High throughput detection of M6P/IGF2R intronic hypermethylation and LOH in ovarian cancer

Cell surface mannose 6-phosphate/insulin-like growth factor II receptors (M6P/IGF2R) bind and target exogenous insulin-like growth factor II (IGF2) to the prelysosomes where it is degraded. Loss of heterozygosity (LOH) for M6P/IGF2R is found in cancers, with mutational inactivation of the remaining allele. We exploited the normal allele-specific differential methylation of the M6P/IGF2R intron 2 CpG island to rapidly evaluate potential LOH in ovarian cancers, since every normal individual is informative. To this end, we developed a method for bisulfite modification of genomic DNA in 96-well format that allows for rapid methylation profiling. We identified ovarian cancers with M6P/IGF2R LOH, but unexpectedly also found frequent abnormal acquisition of methylation on the paternally inherited allele at intron 2. These results demonstrate the utility of our high-throughput method of bisulfite modification for analysis of large sample numbers. They further show that the methylation status of the intron 2 CpG island may be a useful indicator of LOH and biomarker of disease.     

Physical activity and global genomic DNA methylation in a cancer-free population

Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.Key words: DNA methylation, physical activity, peripheral blood     

Physiological regulation of phospholipid methylation alters plasma homocysteine in mice

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.     

Combined hypermethylation of APC and GSTP1 as a molecular marker for prostate cancer: quantitative pyrosequencing analysis

A total of 149 human prostate tissues obtained from our institute were assessed: 52 specimens of benign prostate hyperplasia (BPH) and 97 specimens of prostate cancer (PCa). The methylation status of the genes of Adenomatous polyposis coli (APC) and glutathione-S-transferase-P1 (GSTP1) was analyzed by quantitative pyrosequencing. A methylation score (M score) was calculated to capture the combined methylation level of both genes. The methylation level of each single gene and that of both genes combined was significantly higher in PCa specimens than in BPH (each p < 0.001). The value of APC methylation, GSTP1 methylation, and M score for predicting PCa was measured by the area under the receiver operating characteristic (ROC) curve and reached 0.954, 0.942, and 0.983, respectively. The sensitivity and specificity of the M score in discriminating between PCa and BPH reached 92.8% and 100.0%, respectively. The M score was positively associated with the serum prostate-specific antigen (PSA) level (p trend < 0.001). Our study demonstrates that the quantitative measurement of two methylation markers might drastically improve the ability to discriminate PCa from BPH.     

华南植物园锥栗F-MSAP采样策略及遗传多样性分析

基因组甲基化修饰受环境因素的影响。在以甲基化为代表的表观遗传学研究中,如何减少保存环境对异地采后样品的影响,提高整个实验的准确性和科学性,目前尚未有系统的认知。该研究选取5种常用的采后样品保存方式(液氮冷冻、-20℃冷冻、变色硅胶干燥、密封袋密封、75%酒精浸泡),分别用Wilcoxon signed ranks tests统计分析和UPGMA聚类分析方法,对华南植物园锥栗进行F-MSAP研究,以期找出最佳保存方式。同时,利用正交试验法对F-MSAP体系进行优化,筛选出9对引物(E3-H/M2;E5-H/M2;E6-H/M1;E6-H/M5;E8-H/M1;E8-H/M5;E9-H/M2;E11-H/M5;E14-H/M1),并对不同发育时期的锥栗甲基化水平及遗传多样性进行了论述。结果表明:在锥栗F-MSAP的研究中,Willcoxon signed ranks tests统计分析和UPGMA聚类分析结论一致,密封袋保存效果最佳;成熟叶半甲基化率(27.83%)和总甲基化率(51.13%)高于幼叶(21.35%,45.90%),全甲基化率(23.30%)低于幼叶(24.55%),平均多态位点百分数39.60%,香农信息指数0.207±0.002,表现出较高的甲基化水平和遗传多样性。     

Nuclear DNA changes within Helianthus annuus L.: variations in the amount and methylation of repetitive DNA within homozygous progenies

Complex alterations in the redundancy and methylation of repeated DNA sequences were shown to differentiate the nuclear genome of individuals belonging to single progenies of homozygous plants of the sunflower. DNA was extracted from seedlings obtained from seeds collected at the periphery of flowering heads (P DNA) or from seedlings obtained from seeds collected in their middle (M DNA). Three fractions of repeated sequences were isolated from genomic DNA: a highly repetitive fraction (HR), which reassociates within an equivalent Cot of about 2 × 10^-1, and two medium repetitive fractions (MR1 and MR2) having Cot ranges of about 2 × 10^-1-2 and 2-10², respectively. Denaturation kinetics allowed different sequence families to be recognized within each fraction of repetitive DNA, and showed significant differences in sequence redundancy to occur between P and M DNA, particularly as far as the MR2 fraction is concerned. Most DNA sequence families are more represented in P DNA than in M DNA. However, the redundancy of certain sequences is greater in the latter than in the former. Each repetitive DNA fraction was hybridized to Southern blots of genomic P or M DNA which was digested to completion by three pairs of isoschizomeric restriction endonucleases which are either insensitive or sensitive to the methylation of a cytosine in the recognition site. The results obtained showed that the repetitive DNA of H. annuus is highly methylated. Clear-cut differences in the degree of methylation of P and M DNA were found, and these differences were particularly apparent in the MR2 fraction. It is suggested that alterations in the redundancy of given DNA sequences and changes in their methylation patterns are complementary ways to produce continuous genotypic variability within the species which can be exploited in environmental adaptation.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2     

Effect of Monomethylethanolamine, Dimethylethanolamine, Gangliosides, Isoproterenol, and 2-Hydroxyethylhydrazine on the Conversion of Ethanolamine to Methylated Products by Cultured Chick Brain Neurons

The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10(-8) M and 10(-5) M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10(-6) M and 10(-5) M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 x 10(-5) M propranolol.     

Taxonomic significance of differences in DNA methylation within the 'Mycoplasma mycoides cluster' detected with restriction endonucleases MboI and DpnI

DNA from 22 strains belonging to the ' Mycoplasma mycoides cluster' was tested for methylation of adenine in GATC sequences by its resistance or susceptibility to digestion by the restriction endonucleases MboI , which is inhibited by the methylation, or DpnI , which requires the methylation. Strains of bovine serogroup 7, M. mycoides subsp. mycoides SC, and some M. mycoides subsp. capri strains plus M. capricolum strain Cal Kid lacked the methylation, whereas other strains of M. mycoides subsp. capri and of M. capricolum , plus the F38-like and M. mycoides subsp. mycoides LC strains, possessed it. We conclude that this simple test could provide a valuable criterion in identifying these mycoplamas.     

Genome-wide methylation profiling identifies novel methylated genes in neuroblastoma tumors

Neuroblastoma is a very heterogeneous tumor of childhood. The clinical spectra range from very aggressive metastatic disease to spontaneous regression, even without therapy. Aberrant DNA methylation pattern is a common feature of most cancers. For neuroblastoma, it has been demonstrated both for single genes as well as genome-wide, where a so-called methylator phenotype has been described. Here, we present a study using Illumina 450K methylation arrays on 60 neuroblastoma tumors. We show that aggressive tumors, characterized by International Neuroblastoma Risk Group (INRG) as stage M, are hypermethylated compared to low-grade tumors. On the contrary, INRG stage L tumors display more non-CpG methylation. The genes with the highest number of hypermethylated CpG sites in INRG M tumors are TERT, PCDHGA4, DLX5, and DLX6-AS1. Gene ontology analysis showed a representation of neuronal tumor relevant gene functions among the differentially methylated genes. For validation, we used a set of independent tumors previously analyzed with the Illumina 27K methylation arrays, which confirmed the differentially methylated sites. Top candidate genes with aberrant methylation were analyzed for altered gene expression through the R2 platform (http://r2.amc.nl), and for correlations between methylation and gene expression in a public dataset. Altered expression in nonsurvivors was found for the genes B3GALT4 and KIAA1949, CLIC5, DLX6-AS, TERT, and PIRT, and strongest correlations were found for TRIM36, KIAA0513, and PIRT. Our data indicate that methylation profiling can be used for patient stratification and informs on epigenetically deregulated genes with the potential of increasing our knowledge about the underlying mechanisms of tumor development.     

DNA methylation and Mycoplasma genomes

DNA methylation is one of the many hypotheses proposed to explain the observed deficiency in CpG dinucleotides in a variety of genomes covering a wide taxonomic distribution. Recent studies challenged the methylation hypothesis on empirical grounds. First, it cannot explain why the Mycoplasma genitalium genome exhibits strong CpG deficiency without DNA methylation. Second, it cannot explain the great variation in CpG deficiency between M. genitalium and M. pneumoniae that also does not have CpG-specific methyltransferase genes. I analyzed the genomic sequences of these Mycoplasma species together with the recently sequenced genomes of M. pulmonis, Ureaplasma urealyticum, and Staphylococcus aureus, and found the results fully compatible with the methylation hypothesis. In particular, I present compelling empirical evidence to support the following scenario. The common ancestor of the three Mycoplasma species has CpG-specific methyltransferases, and has evolved strong CpG deficiency as a result of the specific DNA methylation. Subsequently, this ancestral genome diverged into M. pulmonis and the common ancestor of M. pneumoniae and M. genitalium. M. pulmonis has retained methyltransferases and exhibits the strongest CpG deficiency. The common ancestor lost the methyltransferase gene and then diverged into M. genitalium and M. pneumoniae. M. genitalium and M. pneumoniae, after losing methylation activities, began to regain CpG dinucleotides through random mutation. M. genitalium evolved more slowly than M. pneumoniae, gained relatively fewer CpG dinucleotides, and is more CpG-deficient.     

Modifications in major satellite methylation in the nucleus of a two-cell mouse embryo with respect to developmental conditions

A study was conducted of the degree of DNA methylation in the nucleus, in particular, of the major satellite in two-cell mouse embryos developing in the maternal organism, in standard culture medium M16, used for cultivating mouse embryos; and M2 media used for manipulating embryos in air. Two-cell embryo nuclei at 44–46 h after injections of chorionic hormone were investigated. The results are evidence for the dependence of the major satellite’s methylation level on the developmental conditions of embryos. The methylation level of the nuclear DNA was shown to increase with a deterioration of environmental conditions. It was reported that in the case of cultivation in M2 media unsuitable for long cultivation, the DNAís methylation level, the major satellite in particular, was higher compared to other embryo groups. Accordingly, not only a significant number of genes but also sequences of satellite DNA are involved in epigenetic regulation.     

Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells.

The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.     

The MtsA subunit of the methylthiol:coenzyme M methyltransferase of Methanosarcina barkeri catalyses both half-reactions of corrinoid-dependent dimethylsulfide: coenzyme M methyl transfer

Methanogenesis from dimethylsulfide requires the intermediate methylation of coenzyme M. This reaction is catalyzed by a methylthiol:coenzyme M methyltransferase composed of two polypeptides, MtsA (a methylcobalamin:coenzyme M methyltransferase) and MtsB (homologous to a class of corrinoid proteins involved in methanogenesis). Recombinant MtsA was purified and found to be a homodimer that bound one zinc atom per polypeptide, but no corrinoid cofactor. MtsA is an active methylcobalamin:coenzyme M methyltransferase, but also methylates cob(I)alamin with dimethylsulfide, yielding equimolar methylcobalamin and methanethiol in an endergonic reaction with a K(eq) of 5 x 10(-)(4). MtsA and cob(I)alamin mediate dimethylsulfide:coenzyme M methyl transfer in the complete absence of MtsB. Dimethylsulfide inhibited methylcobalamin:coenzyme methyl transfer by MtsA. Inhibition by dimethylsulfide was mixed with respect to methylcobalamin, but competitive with coenzyme M. MtbA, a MtsA homolog participating in coenzyme M methylation with methylamines, was not inhibited by dimethylsulfide and did not catalyze detectable dimethylsulfide:cob(I)alamin methyl transfer. These results are most consistent with a model for the native methylthiol:coenzyme M methyltransferase in which MtsA mediates the methylation of corrinoid bound to MtsB with dimethylsulfide and subsequently demethylates MtsB-bound corrinoid with coenzyme M, possibly employing elements of the same methyltransferase active site for both reactions.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.