Genomic methylation patterns are established by DNA methyltransferases MTases , which catalyze targeted transfers of methyl groups from S-adenosyl-L-methionine SAM, 1 to adenine or cytosine residues. In higher eukaryotes, the cytosine-5 methylation leads to strong and heritable gene silencing 1. Along with DNA demethylation events, whereby 5-methylcytosines mC, 2 are converted back to cytosines 2 , these covalent modifications at the 5 position of cytosine have vital roles in cellular differentiation, parental imprinting and silencing of endogenous retroviruses. Enzymatic transmethylations generally proceed via a direct nucleophilic SN2 attack of a target atom onto the sulfonium-bound methyl group of SAM 3 , 4. DNA cytosinemethyltransferases C5-MTases 5 Scheme 1a , RNA U5-MTases 6 , thymidylate synthases and 5-hydroxymethylcytidylate synthases 7 all use a covalent mechanism for nucleophilic activation of their target pyrimidine residues.
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Genomic methylation patterns are established by DNA methyltransferases MTases , which catalyze targeted transfers of methyl groups from S-adenosyl-L-methionine SAM, 1 to adenine or cytosine residues. In higher eukaryotes, the cytosine-5 methylation leads to strong and heritable gene silencing 1. Along with DNA demethylation events, whereby 5-methylcytosines mC, 2 are converted back to cytosines 2 , these covalent modifications at the 5 position of cytosine have vital roles in cellular differentiation, parental imprinting and silencing of endogenous retroviruses.
Enzymatic transmethylations generally proceed via a direct nucleophilic SN2 attack of a target atom onto the sulfonium-bound methyl group of SAM 3 , 4. DNA cytosinemethyltransferases C5-MTases 5 Scheme 1a , RNA U5-MTases 6 , thymidylate synthases and 5-hydroxymethylcytidylate synthases 7 all use a covalent mechanism for nucleophilic activation of their target pyrimidine residues.
A model system for mechanistic studies of C5 methylation is the HhaI methyltransferase M. This modification is reversed back to unmodified DNA by the enzyme in the absence of the exogenous aldehyde. Modifying reagents are shown in red, C5-MTase and its catalytic moieties are shown in blue, and boxed areas denote species and reactions within the catalytic center of the enzyme. Depending on whether the exocyclic hydroxyl group or the ring 5 position is deprotonated, the release of enzyme yields either the unmodified cytosine and free formaldehyde 3 left or the coupling product 9 right , respectively.
Color coding as in a. Full size image To explore the chemical reactivity of ACI, binary MTase-DNA complexes were prepared and screened against a series of electrophilic compounds such as aldehydes, ketones and electronegatively substituted vinyl derivatives. Notably, analyses of binary M. HhaI-DNA complexes treated with formaldehyde 3 showed a conversion of the target cytosine into a new compound Fig. Additional support for the C5 coupling was obtained from i the absence of 5-H atoms 5 in reaction products Supplementary Fig.
Therefore, we conclude that the M. HhaI-activated cytosine is converted to hmC. Notably, our control experiments showed no detectable formation of N-hydroxymethylated nucleosides Supplementary Fig. HhaI-premethylated or nonspecific was treated in the presence of M. HhaI with formaldehyde or acetaldehyde as described in Supplementary Methods online.
HhaI with formaldehyde, acetaldehyde, SAM or no exogenous reagent control and then enzymatically fragmented to nucleosides and analyzed by reversed-phase HPLC. HhaI with cofactor SAM or formaldehyde and then fragmented with a restriction endonuclease as shown and analyzed by agarose gel electrophoresis. HhaI and formaldehyde step 1 and then treated with M. HhaI and formaldehyde and then treated with M.
HhaI alone and analyzed as in b trace assignments as in d. HhaI and with formaldehyde or acetaldehyde step 1. Modified DNA was then separately incubated with M. HhaI step 2 , fragmented with R. Hin6I and analyzed as in c. Full size image Similar experiments with acetaldehyde 4 , Fig. TLC analyses showed minor amounts of modification products upon treatment with benzyloxyacetaldehyde 7 and betainic aldehyde 8 Supplementary Fig. Altogether, our results demonstrate that the addition reaction is general for short aliphatic aldehydes as a class.
We found the other classes of electrophilic compounds to be inactive. To assess the enzymatic generality of this reaction, a series of commercially available bacterial C5-MTases M. SssI, CG; M. All of the interrogated MTases showed substantial catalytic activity with the aldehydes Supplementary Fig.
The mouse Dnmt1 MTase also followed the trend with minor but detectable formation of the modified product in the presence of formaldehyde Supplementary Fig. Despite variations in the coupling efficiency observed with individual enzymes, the identity of a modified nucleotide was fully dependent on the aldehyde used. On the other hand, the MTase-assisted coupling reactions occurred with high sequence specificity on both short DNA duplexes and large natural substrates Fig.
To our knowledge, this is the first demonstration of wild-type cofactor-dependent enzymes catalyzing an atypical chemical reaction using non-cofactor-like exogenous substrates The reactive aldehydes are not bona fide cofactors of SAM-dependent MTases because they lack an anchor moiety such as adenosyl that would assist in the formation of a discrete, specific complex with the enzyme.
The chemical reaction nucleophilic addition itself is different from the SN2 transfer nucleophilic substitution naturally catalyzed by MTases 3. Normally, aldehydes attack exocyclic amino groups of nucleobases in DNA, which leads to N-hydroxymethyl derivatives and further disubstituted products 14 , These reactions are largely responsible for the cytotoxicity of formaldehyde in vivo 14 , and are exploited for cross-linking of interacting proteins 10 and mapping of unpaired nucleotides 11 , 15 in DNA.
The MTase-directed aldehyde coupling occurs under mild conditions and with high sequence and base specificity. The triple role of an MTase is thus to i recognize a specific DNA sequence, ii present a target nucleobase in the catalytic center and iii covalently activate the C5 position in the base. In crystal structures of the M. These steric factors explain the switch in aldehyde regiospecificity in the presence of C5-MTases from N4 to C5 and suggest that the stereochemistry of the methylation reaction will be followed Scheme 1a.
Consistent with this model, a steric enlargement of the cofactor binding pocket in M. HhaI ref. In genomic DNA, 5-hydroxymethylated pyrimidines hmC and hmU are known to arise via two major pathways. They are incorporated during DNA synthesis to replace the major bases in certain bacteriophages In higher eukaryotes, chemical 19 and enzymatic 18 , 20 , 21 oxidation of mC and thymine is thought to be the sole source of these bases; however, their roles in epigenetic regulation are currently unclear.
The unveiled promiscuity of C5-MTases presents a third possible pathway for the formation of genomic 5-hydroxymethylpyrimidines, as formaldehyde occurs in millimolar concentrations in certain tissues of rats and humans Besides the biological implications, a stable C-C bond generated in the MTase-directed reactions opens new ways for sequence-specific derivatization of DNA. The coupling reactions involving formaldehyde are simple, fast and robust, and are thus suitable for routine laboratory applications; modifications with longer aldehydes can be improved by steric engineering of the enzymes see above.
For example, a mild oxidation to formyl or keto groups would enable a further conjugation with compounds carrying hydrazine or hydroxylamine functions Importantly, because the MTase-directed aldehyde coupling is blocked by prior 5-methylation of the same residue Fig. We also examined whether C5-MTases can promote the reverse reaction—the removal of formaldehyde from hmC. For this, a DNA duplex that contained enzymatically produced hmC residues at the target position was again treated with the same MTase in the absence of the aldehyde.
The amount of hmC was substantially reduced after such treatment, whereas the amount of cytosine was increased Fig. Similar results were obtained with acetaldehyde-modified DNA Fig. These observations demonstrate that C5-MTases catalyze the removal of the coupled aldehyde, thus restoring the original cytosine residue in DNA. The MTase-assisted removal of the C5-bound hydroxymethyl group is noteworthy in light of the fact that the enzymatic methyl transfer is irreversible 4 , 5. The reverse reaction also requires covalent catalysis Fig.
A mechanism for this reaction Scheme 1b can be derived from analogy with the light- and alkali-induced conversions of hmC to cytosine, which occur via corresponding 5,6-dihydrohydroxy derivatives The biological importance of dehydroxymethylation reactions is bolstered by their relevance to the mechanisms of DNA demethylation and repair.
The enzymology of genomic mC demethylation remains elusive and highly debated 2 ; among others, a similar two-step route has been proposed based on the above examples 2 , 20 , 21 and preliminary observations Our findings Fig. Note: Supplementary information and chemical compound information is available on the Nature Chemical Biology website.
CYTOSINE-5-METHYLTRANSFERASES ADD ALDEHYDES TO DNA PDF
We now report the presence of 5-hydroxymethylcytosine 5hmC as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 DNMT1 translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion.
Cytosine-5-methyltransferases add aldehydes to DNA.
Zululabar The coupling reactions involving formaldehyde not bona fide cofactors of SAM-dependent MTases because they lack are simple, fast and robust, and are thus suitable for routine laboratory an anchor moiety such as adenosyl that would assist in the formation applications; modifications with longer aldehydes can be improved by of a discrete, specific complex with the enzyme. Institutional access Recommend FPrime to your librarian or information manager to request an extended free trial for all users at your institution. Here we show that cytosinemethyltransferases catalyze reversible covalent addition of exogenous aliphatic aldehydes to their target residues in DNA, thus yielding corresponding 5-hydroxyalkylcytosines. To our and humans Vilkaitis Institute of Biotechnology for a sample of mouse These steric We also examined whether C5-MTases can promote the reverse factors explain the switch in aldehyde regiospecificity in the presence of reaction—the removal of formaldehyde from hmC. HhaI and with formaldehyde or acetaldehyde step 1.