DNA Methyltransferases 205
responding restriction endonuclease in the case ofprokaryote DNA Mtases.
Mammalian dC Mtases have been shown to be extremely sensitive to
KCI: a concentration of 25 mM giving optimal activity with the majority
of mammalian enzymes.
2.5. Buffer Components
Again for any given enzyme, the buffer that balances optimum rates
with complementary stabilization of the enzyme can only be deter-
mined by a program of empirical experiments. Tris buffers have been
traditionally employed for restriction and modification enzymes, although
there is no apriori reason why this should be the case. Indeed, phosphate
buffers are generally easier to titrate to a given pH and are excellent
buffers at pH 7.4, the pH optimum of many if not all of the DNA Mtases
so far isolated. For convenience, however, the three types of restric-
tion enzyme buffers described by Maniatis et al. (9) are suitable for
most purposes. The buffering agent is usually employed at a concentra-
tion of between 50 and 100 mM and is often supplemented with dithio-
threitol (1 mM) or I~-mercaptoethanol (5 mM) and bovine serum albumin
(100 ktg/mL). The latter reagents are not always necessary and are occa-
sionally inhibitory. If a new Mtase is being isolated, it is best to determine
the enzyme activity in the presence and absence of these reagents.
The reaction is usually carried out at 37°C for between 1 and 2 h and
quenched by the addition of an equal vol of phenol. The reaction vol
should be around 20 ktl-,, although preparative incubations of 200 l.tL
may be required if the DNA is to be analyzed further.
2.6. Experimental Procedures
2.6.1. Materials- 50 mM Tris-HC1, pH 8.0, 100 mM NaC1, 1 mM EDTA.
- Bio-Rad A 0.5M gel.
- 0.3 M Potassium acetate (or ammonium acetate).
- Ethanol.
- [3H-methyl]-S-adenosylmethionine (SAM).
- 100 mM NaCl.
- Assay
The quantitative estimation of DNA Mtase activity can be acheived
in several ways. However, most methods are based on one of two
principles. The first and perhaps the simplest involves the enzyme
- Assay