CYTOSINE-5-METHYLTRANSFERASES ADD ALDEHYDES TO DNA PDF

Saulius Klimasauskas D. Notably, analyses of binary M. HhaI-DNA complexes treated with. All rights reserved. Additional support for the C5 coupling was covalent addition of exogenous aliphatic aldehydes to obtained from i the absence of 5-H atoms5 in reaction products their target residues in DNA, thus yielding corresponding Supplementary Fig.

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Saulius Klimasauskas D. Notably, analyses of binary M. HhaI-DNA complexes treated with. All rights reserved. Additional support for the C5 coupling was covalent addition of exogenous aliphatic aldehydes to obtained from i the absence of 5-H atoms5 in reaction products their target residues in DNA, thus yielding corresponding Supplementary Fig. Such atypical enzymatic reactions 5-methylated cytosine to the formaldehyde treatment Fig. There- with non-cofactor-like substrates open new ways for sequence- fore, we conclude that the M.

HhaI-activated cytosine is converted to specific derivatization of DNA and demonstrate enzymatic hmC. Notably, our control experiments showed no detectable forma- exchange of 5-hydroxymethyl groups on cytosine in support tion of N-hydroxymethylated nucleosides Supplementary Fig. DNA methylation is an important biological mechanism that regulates Similar experiments with acetaldehyde 4, Fig. Genomic methylation patterns are established by DNA In higher eukaryotes, the cytosine-5 methylation a-carbon.

TLC analyses showed minor amounts of modification leads to strong and heritable gene silencing1. Along with DNA products upon treatment with benzyloxyacetaldehyde 7 and betainic demethylation events, whereby 5-methylcytosines mC, 2 are con- aldehyde 8 Supplementary Fig. HhaI, which is consistent with a recently reported DNA Enzymatic transmethylations generally proceed via a direct nucleo- strand cleavage at CAA-modified sites upon piperidine treatment A model Altogether, our results demonstrate that the addition reaction is system for mechanistic studies of C5 methylation is the HhaI general for short aliphatic aldehydes as a class.

We found the other methyltransferase M. HpaII, target site analogs reviewed in ref. SssI, CG; M. AluI, AGCT were examined in a similar 5-H proton with water5 and a slow hydrolytic deamination to afford manner using appropriate DNA substrates12 see Supplementary uracil9 when cofactor is not present. Table 1. The mouse complexes were prepared and screened against a series of electrophilic Dnmt1 MTase also followed the trend with minor but detectable compounds such as aldehydes, ketones and electronegatively substi- formation of the modified product in the presence of formal- tuted vinyl derivatives.

Correspondence should be addressed to S. Received 16 December ; accepted 3 April ; published online 10 May ; doi Color coding as in a. They are incorporated during DNA nucleotide was fully dependent on the aldehyde used. On the other synthesis to replace the major bases in certain bacteriophages In hand, the MTase-assisted coupling reactions occurred with high higher eukaryotes, chemical19 and enzymatic18,20,21 oxidation of mC sequence specificity on both short DNA duplexes and large natural and thymine is thought to be the sole source of these bases; however, substrates Fig.

The unveiled Altogether, our findings reveal a previously unknown ability of promiscuity of C5-MTases presents a third possible pathway for C5-MTases to catalyze the addition of exogenous aliphatic aldehydes the formation of genomic 5-hydroxymethylpyrimidines, as formal- to the C5 position of the target cytosine, thereby yielding nucleobase dehyde occurs in millimolar concentrations in certain tissues of rats derivatives with a-hydroxylated carbon chains Scheme 1a.

To our and humans The reactive aldehydes are derivatization of DNA. 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. The chemical reaction steric engineering of the enzymes see above.

Although the a- nucleophilic addition itself is different from the SN2 transfer nucleo- hydroxyalkyl groups themselves are not sound reporters, they add a philic substitution naturally catalyzed by MTases3.

Normally, alde- unique functionality to DNA analogous to benzylic hydroxyl that can hydes attack exocyclic amino groups of nucleobases in DNA, which be exploited for chemical or enzymatic derivatization. For example, a leads to N-hydroxymethyl derivatives and further disubstituted mild oxidation to formyl or keto groups would enable a further products14, These reactions are largely responsible for the conjugation with compounds carrying hydrazine or hydroxylamine cytotoxicity of formaldehyde in vivo14, and are exploited for cross- functions Alternatively, hmC residues can be enzymatically glucosy- linking of interacting proteins10 and mapping of unpaired nucleo- lated18, thereby permitting selective DNA labeling through application tides11,15 in DNA.

On the other hand, a mild conditions and with high sequence and base specificity. In crystal structures of the M. HhaI by prior 5-methylation of the same residue Fig. 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. For this, a DNA C5-MTases from N4 to C5 and suggest that the stereochemistry of duplex that contained enzymatically produced hmC residues at the the methylation reaction will be followed Scheme 1a.

Consistent with target position was again treated with the same MTase in the absence of this model, a steric enlargement of the cofactor binding pocket in the aldehyde. The amount of hmC was substantially reduced after such M. HhaI ref. Hhal WT Figure 1 Reversible covalent modifications of cytosine with aliphatic aldehydes in the 0. HhaI-premethylated M. HhaI with formaldehyde or acetaldehyde as 0. HhaI with formaldehyde, acetaldehyde, SAM or no exogenous reagent control 0.

HhaI Retention time min 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.

These Education and Science of Lithuania. The reverse reaction also requires covalent catalysis 1. Goll, M.

Morgan, H. A mechanism for this reaction R47—R58 Klimasauskas, S. Trends Biotechnol. Scheme 1b can be derived from analogy with the light- and 4. The biological 5. Wu, J. Bujnicki, J. Nucleic Acids Res. Graves, K. Biochemistry 31, — Cheng, X. Certain DNA repair enzymes can reverse alkylation damage via 9. Zingg, J. Solomon, M. USA 82, — Daujotyte, D. The 36, e57 Roberts, R.

Bornscheuer, U. Wang, M. Kusmierek, J. Biochemistry 21, — Cell 76, — Lukinavicius, G. Gommers-Ampt, J. Note: Supplementary information and chemical compound information is available on Privat, E.

Tahiliani, M. Science published online, doi Kriaucionis, S. Heck, H. Sowers Loma Linda University for the gift of an hmC-containing Prescher, J. Vilkaitis Institute of Biotechnology for a sample of mouse Chittaboina, S. Tetrahedron Lett. Dnmt1, and M. Thanks are due to L.

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CYTOSINE-5-METHYLTRANSFERASES ADD ALDEHYDES TO DNA PDF

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.

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Cytosine-5-methyltransferases add aldehydes to DNA.

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.

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Cytosine-5-methyltransferases add aldehydes to DNA

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