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The mechanism employed by DNA (cytosine-5)-methyltransferases (m5C MTases), as originally proposed by Santi et al. and later modified, is presented in the Figure above(A). The reaction is initiated by covalent attack of a cysteine thiolate on C6 of the substrate cytosine (Step 1). In order to avoid the formation of a high-energy carbanion, it has been proposed that the thiolate attack at C6 is accompanied by protonation at N3 by an enzyme-derived acid. The resulting structure, termed an enamine, attacks the methyl group of S-adenosyl-L-methionine, transferring it to C5 (Step 2). Abstraction of the proton at C5 again yields an enamine (Step 3), which undergoes conjugate elimination to yield the product, 5-methyl-dC (Step 4). Early on, it was argued that a mechanism could only be attained at the expense of substantial distortion of DNA structure, because the required trajectory for attack of the thiolate on C6 was blocked by the DNA backbone, and the trajectory for methyl delivery at C5 was occluded by the neighboring DNA bases (B). A curious feature of this mechanism is that it would also seem to require some sort of strand separation event, in order to permit access of the enzyme-derived acid to N3 (which is ordinarily involved in Watson-Crick base-pairing). The finding that an oligonucleotide having the two strands tied together by a disulfide cross-link was still a substrate for the DNA methyltransferase M.Hae III suggested the strand-separation event is highly localized, involving perhaps only the substrate cytosine. Based on the covalent catalysis mechanism, Santi and co-workers designed the inhibitor 5-fluoro-2'-deoxycytidine (FdC), in which the 5H of cytosine is replaced by a fluorine. This inhibitor blocks progression through Step 3 of the mechanism, presumably because abstraction of F+ is virtually impossible at physiological conditions; indeed, FdC has been shown to form covalent complex with a wide range of bacterial and mammalian m5C MTases. However, H and F bonded to C possess a similar van der Waals radius, the trapped intermediate thus formed is expected to resemble closely the native intermediate formed with C. |
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DNA methylation has come of age. For more than a decade, it has been
known that eukaryotes tag their DNA by the covalent addition of a methyl group to cytosine
residues; however, until very recently, the functional significance of this modification
has remained on a precariously speculative footing. A series of discoveries over the last
few years has thrust DNA methylation firmly into the mainstream of biology and medicine,
thereby invigorating the field with a firmly established sense of mission. Fragile-X
syndrome, the leading cause of inherited mental retardation, has been traced to expansion
and abnormal methylation of a triplet repeat, through which transcription of the FMR-1
gene becomes silenced. The finding that cells can be transformed by constitutive
overexpression of a eukaryotic DNA methyltransferase enzyme, taken together with apparent
instances of tumor suppressor inactivation through promoter methylation, have strengthened
the widely suspected connection between aberrant DNA methylation and cancer. Much
excitement has revolved around the role of DNA methylation in genomic imprinting, a
phenomenon in which parental alleles of the same gene are expressed at unequal dosage.
Abnormal expression of imprinted loci is now implicated in several human disorders, and
more instances seem likely to be discovered. The entire field of DNA methylation, with its
foundations resting largely on the strength of correlative data, took both a collective
breath of relief and a bold step forward with the direct demonstration that the DNA
methyltransferase gene is essential for development in mice. A molecular mechanism for
spatio-temporal coupling of DNA replication and methylation has been suggested by the
finding that DNA methyltransferase interacts directly with the replication apparatus.
