Methods in Molecular Biology • 16 Enzymes of Molecular Biology

202 Hornby

NH2 NH2

0 ENZ

0 I ENZYME I

R R

(a) (b)

8AM NH2

R

Icl

NH2

R

Id)

Fig. 1. Proposed mechanism of action of 5' cytosine-specific DNA methyltrans-
ferases based on the studies of thymidilate synthase and Hha I DNA methyltransferase.

cient to render a sequence refractory to endonucleolytic cleavage by
a restriction enzyme of the same specificity (see Chapter 8, Sections 2.4.1.
and 3.7.). This is the molecular basis of restriction and modification. In
mammalian cells, cytosine methylation has been implicated in the regu-
lation of gene transcription and in the inactivation of the X chromosome,
although the molecular details of these processes remain unresolved (5).
The reaction catalyzed by all DNA Mtases is shown below. SAM
invariably acts as the methyl donor, and the natural acceptor is chro-
mosomal DNA, but in vitro, plasmid DNA, phage DNA, or an appro-
priate oligonucleotide duplex can also act as methyl acceptors.

DNA + SAM ~ MeDNA + SAH

The products of the reaction are methylated DNA and S-adenosylhomo-
cysteine (SAH). The site of modification is enzyme-specific. Little is
known about the mechanism of dA methylation, whereas studies on
the mechanistically related enzyme thymidilate synthase have led to
the proposed scheme for dC methylation shown in Fig. 1 (6). Interest-
ingly, a catalytic cysteine flanked on the N-terminal side by a proline