Restriction Enzymes 167tion (e.g., BamHI, BstNI). Another class of restriction enzymes (e.g.,
CfuI, DpnI) only cleaves the sequence Gm6ATC when modified by the
dam methylase or exclusively cuts methylated dcm sites, cmSCWGG
(e.g., ApyI). The sensitivity toward site-specific methylation also may
prevent cleavage of eukaryotic DNA whose cytosine residues may be
methylated in CG or CNG sequences. Whereas HpalI and SmaI are
inhibited by m5CG, MspI and XmaI are not influenced by this type of
methylation. The inability of some restriction enzymes to cleave
methylated DNA can be utilized to obtain large fragments even with
enzymes recognizing hexanucleotide palindromes. The sensitivity of
the commercially available restriction enzymes toward different types
of methylation is summarized in Tables 5, 6, and 7.
To avoid site-specific dam and/or dcm methylation of recognition
sites in E. coli vectors, it is necessary to propagate the vectors in strains
lacking one or both of the methyltransferase systems (e.g., E. coli GM 1674,
which has the genotype dam-dcm-). It must be taken into account,
however, that E. coli strains, deficient in dam or dcm methylation,
have elevated rates of mutation.
Site-specific methylation in vitro, using Type II modification enzymes
or the dam or dcm methyltransferases, can be used to block the subset
of restriction sites that overlap with the methylated sequence (Table
6). Therefore, the MspI methyltransferase (CCGG) can block the cleav-
age of DNAby the restriction endonuclease BamHI (GGATCC) in the
sequences GGATCCGG and CCGGATCC. For restriction endonu-
cleases recognizing degenerate sequences, a new specificity can result.
For example, HinclI recognizes the sequence GTYRAC. The TaqI
methyltransferase inhibits the cleavage of GTCGAC by modifying
TCGA. Thus, the specificity of HinclI is reduced to GTYAAC, when
the DNA has been methylated by MoTaqI.
DNA methylation by Type II modification enzymes can be blocked
by prior methylation with other methyltransferases. If the first methy-
lation does not inhibit the cleavage with the corresponding endonu-
clease, one can selectively cleave those recognition sites where the
second methylation was blocked. For example, methylation with MoMspI
prevents methylation with M°BamHI at GGATCCCGG sequences.
These sequences are cut by BamHI, whereas all other recognition sites
for this enzyme are methylated by its corresponding methylase (see
footnote d in Table 6).