DNA Methyltransferases 203residue is found in all of the pyrimidine-specific Mtases, which modify
at a carbon atom so far characterized. Indeed, amino acid sequences of
the dC DNA Mtases from mouse (7), human (Andrews, P., Burton D.
R., and Hornby, D. P., unpublished results), and a variety of bacteria
(8), although otherwise quite dissimilar, share a pro-cys dipeptide
along with a putative specificity determining sequence toward the C-
terminus of the polypeptide.
It has been suggested (6) that the first stage of C-5, dC methylation
involves the nucleophilic attack by the cysteine-sulfhydryl group at
the C-6 position of the heterocycle resulting in the transient formation
of a covalent enzyme:nucleic acid adduct. Subsequent abstraction of
a proton at the C-5 position following the addition of the activated
methyl group of SAM leads to the formation of 5-methyldC with the
concomitant release of the enzyme (see Fig. 1).- Enzyme Requirements
2.1. S-Adenosylmethionine (SAM)
Storage of this substrate is particularly important, since the half-life
of SAM at pH 7.5 and 37°C is around 20 h. Therefore, SAM should be
prepared immediately prior to assay, or if prolonged storage is desired,
the solution should be made 5 mM with respect to sulfuric acid, 10%
with respect to ethanol, and at a SAM concentration sufficient to faci-
litate subsequent dilution into a well-buffered assay mixture (e.g.,
between 1 and 10 mM). In the latter context, [3H-methyl] SAM is
supplied by the manufacturers in dilute sulfuric acid. In general, the
apparent K m for SAM for those enzymes that have been purified and
kinetically characterized lies between 1 and 100 ~/, and therefore, a
final concentration of 100 JaM of the substrate in the reaction incuba-
tion should be saturating. Of course, there may be instances where the
latter concentration is either subsaturating (K m > 50 ktM) or inhibitory
(substrate inhibition), but this will only emerge following a detailed
investigation of individual Mtase enzymes.
2.2. DNA
In order to assay the activity of a DNA Mtase during a purification
protocol, [3H-methyl] SAM is often employed as the methyl donor. In
this way, a sensitive estimation of methyltransfer can be obtained by
liquid scintillation counting. Alternatively, methylation of a DNA