206 Hornbycatalyzed transfer oftritiated methyl groups to DNA from [3H-methyl]
SAM followed by separation of the reaction components either by
selective precipitation or by chromatography. The tritiated DNA pro-
duced in the incubation is subsequently estimated by liquid scintil-
lation counting.
In the tritium transfer method, the DNA Mtase enzyme is incubated
with the appropriate substrates, and the reaction mixture quenched with
phenol. The tritiated DNAcan then be isolated by chromatography through
a Pasteur pipet-sized column of Bio-Rad A0.5M gel-filtration medium (or
similar). Typically, the reaction mixture is loaded onto the column and
eluted with a buffer containing 50 mMTris-HC1, pH 8, 100 mM NaC1, and
1 mM EDTA. The DNA elutes well in advance of the unreacted SAM.
Alternatively, the DNA can be precipitated with 3 vol of cold ethanol in
the presence of 0.3M potassium (or ammonium) acetate. If precipitation
is used, care should be taken not to "carry over" any unreacted SAM.
Moreover, stringent controls in the absence of individual reaction compo-
nents should be included in order to verify the assay results. This is par-
ticularly important in assays of crude extracts, which probably contain
protein and RNA Mtases.
A different strategy for estimating DNA Mtase activity involves the
use of DNA fragments containing specific enzyme recognition sites.
This approach is particularly convenient in assaying the DNA Mtases
of bacteria. In one method, a DNA fragment containing the methyla-
tion site of, for example, EcoRI Mtase is incubated with the enzyme
in the presence of unlabeled SAM, and the modified DNA so produced
is extracted with phenol and precipitated with ethanol. The DNA is sub-
sequently incubated with EcoRI restriction enzyme. The extent of methy-
lation can be determined by the extent of resistance to endonuclease
digestion. This approach can be made highly sensitive by incorporating
radioactively labeled DNAinto the reaction mixture. The DNA fragments
can either be resolved by gel electrophoresis or by HPLC.
- Applications of DNA Methyltransferases
DNA Mtases have been incorporated into a number of molecular
biology protocols and generally bring a greater degree of flexibility to
molecular cloning strategies. Some of the uses of DNA MTases are
outlined in the following sections.