Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.     

Methylation of slipped duplexes, snapbacks and cruciforms by human DNA(cytosine-5)methyltransferase.

When human DNA(cytosine-5)methyltransferase was used to methylate a series of snapback oligodeoxy-nucleotides of differing stem lengths, each containing a centrally located CG dinucleotide recognition site, the enzyme required a minimum of 22 base pairs in the stem for maximum activity. Extrahelical cytosines in slipped duplexes that were 30 base pairs in length acted as effective methyl acceptors and were more rapidly methylated than cytosines that were Watson-Crick paired. Duplexes containing hairpins of CCG repeats in cruciform structures in which the enzyme recognition sequence was disrupted by a C.C mispair were also more rapidly methylated than control Watson-Crick-paired duplexes. Since enzymes have higher affinities for their transition states than for their substrates, the results with extrahelical and mispaired cytosines suggest that these structures can be viewed as analogs of the transition state intermediates produced during catalysis by methyltransferases.     

Methylation inhibitors can increase the rate of cytosine deamination by (cytosine-5)-DNA methyltransferase.

The target cytosines of (cytosine-5)-DNA methyltransferases in prokaryotic and eukaryotic DNA show increased rates of C-->T transition mutations compared to non-target cytosines. These mutations are induced either by the spontaneous deamination of 5-mC-->T generating inefficiently repaired G:T rather than G:U mismatches, or by the enzyme-induced C-->U deamination which occurs under conditions of reduced levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). We tested whether various inhibitors of (cytosine-5)-DNA methyltransferases analogous to AdoMet and AdoHcy would affect the rate of enzyme-induced deamination of the target cytosine by M.HpaII and M.SssI. Interestingly, we found two compounds, sinefungin and 5'-amino-5'-deoxyadenosine, that increased the rate of deamination 10(3)-fold in the presence and 10(4)-fold in the absence of AdoMet and AdoHcy. We have therefore identified the first mutagenic compounds specific for the target sites of (cytosine-5)-DNA methyltransferases. A number of analogs of AdoMet and AdoHcy have been considered as possible antiviral, anticancer, antifungal and antiparasitic agents. Our findings show that chemotherapeutic agents with affinities to the cofactor binding pocket of (cytosine-5)-DNA methyltransferase should be tested for their potential mutagenic effects.     

Human DNA (cytosine-5)methyltransferase selectively methylates duplex DNA containing mispairs.

The presence of the C.C mispair in a defined duplex oligodeoxynucleotide enhanced its capacity to serve as a substrate for highly purified human DNA methyltransferase. Analysis of tritiated reaction products showed that the C.C mispair acted as a "methylation acceptor" in that it was itself rapidly methylated. The m5C.G base pair also enhanced the capacity of the oligodeoxynucleotide to serve as a substrate for the enzyme. However, this complementary base pair was found to act as a "methylation director". That is, the presence of the m5C in one strand induced the enzyme to rapidly methylate at the cytosine residue on the opposite strand in an adjacent C.G base pair.     

Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.     

Allosteric activator domain of maintenance human DNA (cytosine-5) methyltransferase and its role in methylation spreading

The human maintenance DNA (cytosine-5) methyltransferase (hDNMT1) consists of a large N-terminal regulatory domain fused to a catalytic C-terminal domain by randomly repeated Gly-Lys dipeptides. Several N-terminal deletion mutants of hDNMT1 were made, purified, and tested for substrate specificity. Deletion mutants lacking 121, 501, 540, or 580 amino acids from the N-terminus still functioned as DNA methyltransferases, methylated CG sequences, and preferred hemimethylated to unmethylated DNA, as did the full-length hDNMT1. Methylated DNA stimulated methylation spreading on unmethylated CpG sequences for the full-length and the 121 amino acid deletion hDNMT1 equally well but not for the mutants lacking 501, 540, or 580 amino acids, indicating the presence of an allosteric activation determinant between amino acids 121 and 501. Peptides from the N- and C-termini bound methylated DNA independently. Point mutation analysis within the allosteric region revealed that amino acids 284-287 (KKHR) were involved in methylated DNA-mediated allosteric activation. Allosteric activation was reduced in the double point mutant enzymes D25 (K284A and K285A) and D12 (H286A and R287A). Retinoblastoma gene product (Rb), a negative regulator of DNA methylation, bound to the allosteric site of hDNMT1 and inhibited methylation, suggesting Rb may regulate methylation spreading.     

A G4-DNA/B-DNA junction at codon 12 of c-Ha-ras is actively and asymmetrically methylated by DNA(cytosine-5)methyltransferase

Oligodeoxynucleotides spanning codon 12 of the human c-Ha-ras gene were found to be exceptionally good substrates for de novo methylation by human DNA(cytosine-5)methyltransferase. In the complex formed by two complementary 30mers, only the C-rich strand was methylated by the enzyme. Guanines at the 3' end of the G-rich strand of the complex could not be completely modified by dimethyl sulfate [corrected] suggesting tetrameric bonding at these G-residues. An eight-stranded structure, composed of four duplex DNAs at one end, joined to a G4-DNA segment at the other with the junction between the two DNA forms at codon 12, can account for our results.     

Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus

DNA (cytosine-5) methylation represents one of the most widely used mechanisms of enduring cellular memory. Stable patterns of DNA methylation are established during development, resulting in creation of persisting cellular phenotypes. There is growing evidence that the nervous system has co-opted a number of cellular mechanisms used during development to subserve the formation of long term memory. In this study, we examined the role DNA (cytosine-5) methyltransferase (DNMT) activity might play in regulating the induction of synaptic plasticity. We found that the DNA within promoters for reelin and brain-derived neurotrophic factor, genes implicated in the induction of synaptic plasticity in the adult hippocampus, exhibited rapid and dramatic changes in cytosine methylation when DNMT activity was inhibited. Moreover, zebularine and 5-aza-2-deoxycytidine, inhibitors of DNMT activity, blocked the induction of long term potentiation at Schaffer collateral synapses. Activation of protein kinase C in the hippocampus decreased reelin promoter methylation and increased DNMT3A gene expression. Interestingly, DNMT activity is required for protein kinase C-induced increases in histone H3 acetylation. Considered together, these results suggest that DNMT activity is dynamically regulated in the adult nervous system and that DNMT may play a role in regulating the induction of synaptic plasticity in the mature CNS.     

Methods for the design and analysis of oligodeoxynucleotide-based DNA (cytosine-5) methyltransferase inhibitors

Several second-generation inhibitors of DNA (cytosine-5) methyltransferases based on studies of modified synthetic oligodeoxynucleoides have been described. As an aid to studies of these inhibitors, we present an electronic structure-based algorithm that can be used as a method for predicting the nature of the expected inhibition by any noncytosine nucleotide target. Targeting by the major human enzyme (hDnmt1) is governed by the presence of a three-nucleotide motif. In hemimethylated DNA, this motif consists of a 5-methylcytosine targeting signal that causes the enzyme to probe the opposite strand for a normally paired guanosine or inosine residue and attempt to methylate the residue 5' to that site. As a demonstration of the method, we apply these rules to the design and characterization of a novel oligodeoxynucleotide inhibitor of hDnmt1. This inhibitor takes advantage of the three-nucleotide recognition motif characteristic of hDnmt1 and shows that the enzyme is inhibited in vitro by non-CG methylation which targets the enzyme to normally basepaired but unproductive nucleotides such as dG, dA, and dT. Kinetic analysis at constant S-adenosyl-L-methionine concentration shows that representative inhibitory oligodeoxynucleotides are best viewed as weakly productive components of systems containing two DNA substrates. This model suggests that the most effective inhibitors are those with very low apparent Vmax and very low Km values. Oligodeoxynucleotides containing mispaired and unproductive targets such as dG, dA, dT, and dU are also inhibitory as secondary substrates for the human enzyme. Biologically, fail-safe mechanisms identified by the ab initio approach appear to be active in preventing potentially mutagenic deamination of dihydrocytosine and enzymatic methylation of dU.     

The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate

The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.     

Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC.

A novel gene encoding a cytosine-5-DNA methyltransferase recognizing the dinucleotide GpC was cloned from Chlorella virus NYs-1 and expressed in both Escherichia coli and Saccharomyces cerevisiae . The gene was sequenced and a predicted polypeptide of 362 amino acids with a molecular weight of 41.903 kDa was identified. The protein contains several amino acid motifs with high similarity to those of other known 5-methylcytosine-forming methyltransferases. In addition, this enzyme, named M. Cvi PI, shares 66% identity and 76% similarity with M. Cvi JI, the only other cytosine-5-DNA methyltransferase cloned from a Chlorella virus. The short, frequently occurring recognition sequence of the new methyltransferase will be very useful for in vivo chromatin structure studies in both yeast and higher organisms.     

Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase

M13 DNAs in which carbon 5 of each deoxycytidine residue in one strand is replaced with a bulky group are very good substrates for human DNA (cytosine-5) methyltransferase. Rate enhancements of up to 35 fold are obtained depending on the size of the moiety at C-5. The enzyme appears optimally suited to sense a methyl group in one strand at this position. Alkaline density gradient analyses of the distribution of methyl groups applied to 5-BrdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate the unsubstituted strand.     

Effect of interaction between 5-azacytidine and DNA (cytosine-5) methyltransferase on C-to-G and C-to-T mutations in Escherichia coli.

The purpose of this study was to determine the effect of the Dcm cytosine methyltransferase on 5-azacytidine (5-azaC) mutagenesis in Escherichia coli. We used a Lac reversion assay to measure C-to-G and C-to-T mutations at a single, methylatable cytosine in the lacZ gene, in the presence and absence of Dcm. C-to-G mutations are stimulated by 5-azaC but are largely independent of Dcm. In contrast, C-to-T mutations are not stimulated by 5-azaC in either wild type or dcm cells. However, in cells which contain Dcm but are defective in very short patch repair, the normally high frequency of spontaneous C-to-T mutations is decreased by the analog in a dose-dependent manner.