Cutting and joining DNA molecules - Principles of Gene Manipulation - page 33

A methyltransferase, M. Sss I, that methylates the
dinucleotide CpG (Nur et al. 1985) has been isolated
from Spiroplasma . This enzyme can be used to modify
in vitro restriction endonuclease target sites which
contain the CG sequence. Some of the target
sequences modified in this way will be resistant to
endonuclease cleavage, while others will remain
sensitive. For example, if the sequence CCGG is
modified with Sss I, it will be resistant to Hpa II but
sensitive to Msp I. Since 90% of the methyl groups in
the genomic DNA of many animals, including verte-
brates and echinoderms, occur as 5-methylcytosine
in the sequence CG, M. Sss can be used to imprint
DNA from other sources with a vertebrate pattern.

The second reason these methylases are of inter-
est is that the modification state of plasmid DNA can
affect the frequency of transformation in special situ-
ations. Transformation efficiency will be reduced when
Dam-modified plasmid DNA is introduced into Dam −
E. coli or Dam- or Dcm-modified DNA is introduced into
other species (Russell & Zinder 1987). When DNA is
to be moved from E. coli to another species it is best to
use a strain lacking the Dam and Dcm methylases.
As will be seen later, it is difficult to stably clone
DNA that contains short, direct repetitive sequences.
Deletion of the repeat units occurs quickly, even
when the host strain is deficient in recombination.
However, the deletion mechanism appears to involve
Dam methylation, for it does not occur in dam
mutants (Troester et al. 2000).

The Dam and Dcm methylases of E. coli

Most laboratory strains of E. coli contain three site-
specific DNA methylases. The methylase encoded
by the dam gene transfers a methyl group from
S -adenosylmethionine to the N 6 position of the
adenine residue in the sequence GATC. The methyl-
ase encoded by the dcm gene (the Dcm methylase,
previously called the Mec methylase) modifies the
internal cytosine residues in the sequences CCAGG
and CCTGG at the C 5 position (Marinus et al. 1983).
In DNA in which the GC content is 50%, the sites
for these two methylases occur, on average, every
256 -512 bp. The third methylase is the enzyme
M. Eco KI but the sites for this enzyme are much rarer
and occur about once every 8 kb.
These enzymes are of interest for two reasons.
First, some or all of the sites for a restriction en-
donuclease may be resistant to cleavage when
isolated from strains expressing the Dcm or Dam
methylases. This occurs when a particular base in
the recognition site of a restriction endonuclease
is methylated. The relevant base may be methylated
by one of the E. coli methylases if the methylase
recognition site overlaps the endonuclease recog-
nition site. For example, DNA isolated from Dam +
E. coli is completely resistant to cleavage by Mbo I, but
not Sau 3AI, both of which recognize the sequence
GATC. Similarly, DNA from a Dcm + strain will be
cleaved by Bst NI but not by Eco RII, even though
both recognize the sequence CCATGG. It is worth
noting that most cloning strains of E. coli are Dam +
Dcm + but double mutants are available (Marinus
et al. 1983).

The importance of eliminating
restriction systems in E. coli strains used
as hosts for recombinant molecules

If foreign DNA is introduced into an E. coli host it
may be attacked by restriction systems active in the
host cell. An important feature of these systems is
that the fate of the incoming DNA in the restrictive
host depends not only on the sequence of the DNA
but also upon its history: the DNA sequence may or
may not be restricted, depending upon its source
immediately prior to transforming the E. coli host
strain. As we have seen, post-replication modifications
of the DNA, usually in the form of methylation of par-
ticular adenine or cytosine residues in the target sequ-
ence, protect against cognate restriction systems but
not, in general, against different restriction systems.
Because restriction provides a natural defence
against invasion by foreign DNA, it is usual to
employ a K restriction-deficient E. coli K12 strain
as a host in transformation with newly created
recombinant molecules. Thus where, for example,
mammalian DNA has been ligated into a plasmid
vector, transformation of the Eco K restriction-
deficient host eliminates the possibility that the
incoming sequence will be restricted, even if the
mammalian sequence contains an unmodified Eco K
target site. If the host happens to be Eco K restriction-
deficient but Eco K modification- proficient , propagation
on the host will confer modification methylation and
hence allow subsequent propagation of the recom-
binant in Eco K restriction-proficient strains, if desired.