Reviving a dead enzyme: cytosine deaminations promoted by an inactive DNA methyltransferase and an S-adenosylmethionine analogue

The enzymes that transfer a methyl group to C5 of cytosine within specific sequences (C5 Mtases) deaminate the target cytosine to uracil if the methyl donor S-adenosylmethionine (SAM) is omitted from the reaction. Recently, it was shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI and M.MspI is enhanced in the presence of several analogues of SAM, and a mechanism for this analogue-promoted deamination was proposed. According to this mechanism, the analogues protonate C5 of the target cytosine, creating a dihydrocytosine intermediate that is susceptible to deamination. We show here that one of these analogues, 5'-aminoadenosine (AA), enhances cytosine deamination by the Mtase M. EcoRII, but it does so without enhancing protonation of C5. Further, we show that uracil is an intermediate in the mutational pathway and propose an alternate mechanism for the analogue-promoted deamination. The new mechanism involves a facilitated water attack at C4 but does not require attack at C6 by the enzyme. The latter feature of the mechanism was tested by using M.EcoRII mutants defective in the nucleophilic attack at C6 in the deamination assay. We find that although these proteins are defective in methyl transfer and cytosine deamination, they cause cytosine deaminations in the presence of AA in the reaction. Our results point to a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer methyl groups to N(4) of cytosine. Further, they provide an unusual example where a coenzyme activates an otherwise "dead" enzyme to perform catalysis by a new reaction pathway.     

Homology Modeling of the CG-specific DNA Methyltransferase SssI and its Complexes with DNA and AdoHcy

Abstract

Prokaryotic DNA methyltransferase M. SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed “FRankenstein's monster” approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.     

Homology modeling of the CG-specific DNA methyltransferase SssI and its complexes with DNA and AdoHcy

Prokaryotic DNA methyltransferase M.SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed "FRankenstein's monster" approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.     

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 ± 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R²) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5′-CG-3′ site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.     

The tendency to recreate ancestral CG dinucleotides in the human genome

Background  

The CG dinucleotides are known to be deficient in the human genome, due to a high mutation rate from 5-methylated CG to TG and its complementary pair CA. Meanwhile, many cellular functions rely on these CG dinucleotides, such as gene expression controlled by cytosine methylation status. Thus, CG dinucleotides that provide essential functional substrates should be retained in genomes. How these two conflicting processes regarding the fate of CG dinucleotides - i.e., high mutation rate destroying CG dinucleotides, vs. functional processes that require their preservation remains an unsolved question.     

HpaII methyltransferase is mutagenic in Escherichia coli.

A genetic reversion assay to study C-to-T mutations within CG sites in DNA is described. It was used to demonstrate that the presence of HpaII methyltransferase (MTase) in Escherichia coli causes a substantial increase in C-to-T mutations at CG sites. This is similar to the known mutagenic effects of E. coli MTase Dcm within its own recognition sequence. With this genetic system, a homolog of an E. coli DNA repair gene in Haemophilus parainfluenzae was tested for antimutagenic activity. Unexpectedly, the homolog was found to have little effect on the reversion frequency. The system was also used to show that HpaII and SssI MTases can convert cytosine to uracil in vitro. These studies define 5-methylcytosine as an intrinsic mutagen and further elaborate the mutagenic potential of cytosine MTases.     

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5''-CpG-3'' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.¹ and Clark et al.², methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA

5-Methylcytosine residues in DNA underwent deamination at high temperatures. Furthemore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [¹⁴C]5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [¹⁴C]5-methylcytosine residues. Upon incubation at 95°C in a physiological buffer or at 60°C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those of cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.     

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are ‘hotspots’ for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3′ and 5′ of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.     

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.     

Purification and site-directed mutagenesis of DNA methyltransferase SssI

Prokaryotic DNA methyltransferase SssI (M.SssI) methylates C5 position of cytosine residue in CpG sequences. To obtain functionally active M.SssI and its mutants as His6-tagged proteins, bacterial strains have been produced. To test a possible role of Ser300 in recognition of CpG site by this enzyme, M.SssI mutants containing Ser300 replacements with Gly or Pro have been obtained. These replacements have practically no effect on DNA binding and methylation by M.SssI except small disturbance of DNA binding affinity in the case of S300P mutant. It indicates that there are no interactions of both the side chain and, probably, the main chain of Ser300 with DNA. A replacement of highly conserved Va1188 residue with Ala has been performed. Vall88 may participate in the stabilization of the flipped target cytosine during reaction. The replacement results in a 5-fold decrease of dissociation constant of the enzyme-substrate complex and a 2-fold decrease of initial velocity of DNA methylation. Though there are no noticeable differences in the functioning of the mutant in comparison with the wild-type enzyme, the formation of contact between Val 188 and cytosine could not be excluded. In the case of V 188A mutant the contact may be probably formed between Ala and cytosine residue.     

Distribution of CWG and CCWGG in the Human Genome

Expression of the bacterial CG methyltransferase M?HhaI in mammalian cells appears to generate significant biological effects, while biological effects of the expression of the non-CG methyltransferase M?EcoRII in human cells have not been detected. The association of cytosine methylation with the CG site in mammals is also associated with clustering of CG sites near 5´ control regions (CG-islands) of human genes. Moreover spontaneous deamination of 5-methylcytosine at these sites is thought to lead to the well known deficiency of CG sites in genomes where endogenous CG methyltransferases are expressed. Since these associations are generally taken to imply a biological function for the CG dinucleotide that is associated with its selective methylation by endogenous DNA methylation systems, we have asked whether or not CWG or CCWGG sites are clustered in regions flanking human genes and whether or not an overall deficiency of CWG or CCWGG occurs in the human genome. Using build 36.1, of the human genome, we inspected the regions flanking the 28,501 well known gene loci in the human genome. Our analysis confirmed the expected clustering of CG sites near the 5´ region of known genes and open reading frames. In contrast to the CG site, neither the CWG site nor the CCWGG site recognized by the bacterial methyltransferase M?EcoRII were clustered in any particular region near known genes and open reading frames. Moreover, neither the CCWGG nor the CWG site was depleted in the human genome, again in sharp contrast to the known genomic deficiency of CpG sites. Our findings suggest that in contrast to CG site recognition, human cytosine methyltransferases recognize CWG and CCWGG only at very low frequency if at all.     

Purification and some properties of cytosine deaminase from Salmonella typhimurium

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogenous. The molecular weight of cytosine deaminase was determined to be approx. 230 000 containing four identical subunits with each subunit having a molecular weight of 54 000. Cytosine deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50°C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The K_m and V values for cytosine were 0.74 mM and 47.16 μmole/min, respectively. As effectors of enzyme activity, PP_i stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171–1174).     

The structure of Escherichia coli cytosine deaminase

Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (αβ)₈ barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent.     

Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations

Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor, S-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM. Escherichia coli SAM levels were lowered by reducing SAM synthetase activity via the introduction of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. The metK84 strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible P(BAD) promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.     

High frequency mutagenesis by a DNA methyltransferase.

HpaII methylase (M. HpaII), an example of a DNA (cytosine-5)-methyltransferase, was found to induce directly a high frequency of C-->U transition mutations in double-stranded DNA. A mutant pSV2-neo plasmid, constructed with an inactivating T-->C transition mutation creating a CCGG site, was incubated with M. HpaII in the absence of S-adenosylmethionine (SAM). This caused an approximately 10(4)-fold increase in the rate of reversion when the mutant neo plasmid was transformed into bacteria lacking uracil-DNA glycosylase. The mutation frequency was very sensitive to SAM concentration and was reduced to background when the concentration of the methyl donor exceeded 300 nM. The data support current models for the formation of a covalent complex between the methyltransferase and cytosine. They also suggest that the occurrence of mutational hot spots at CpG sites may not always be due to spontaneous deamination of 5-methylcytosine, but might also be initiated by enzymatic deamination of cytosine and proceed through a C-->U-->T pathway.     

miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism

Methylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively. Two thymine glycosylases are believed to reduce the impact of 5-methylcytosine deamination. G/T mismatch-specific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 can both excise uracil or thymine at U·G and T·G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and methyl-CpG binding domain protein 4. Our results demonstrate (1) that both Tdg and Dnmt3b are colocalized to heterochromatin and (2) reduction of T·G mismatch repair efficiency upon loss of DNA methyltransferase expression, as well as a requirement for an RNA component for correct T·G mismatch repair.     

Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

Several reports suggest that C^mCWGG methylation tends not to co-exist with ^mCG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII–GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C^mCWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C^mCWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C^mCWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C^mCWGG in its promoter. Kinetic studies suggested that an adjacent C^mCWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C^mCWGG methylation does not exert a significant effect on CG methylation in human kidney cells.