Restriction Enzymes 1212.4.1. Methylation
Methylation of critical residues of the recognition sequence in gen-
eral prevents a specific interaction between restriction enzymes and
DNA. In vivo, modification enzymes protect the host DNA through
methylation against cleavage by the corresponding restriction enzyme.
Methylation usually occurs at the amino group at position 6 of an adenine
residue (m6A) or at position 5 of a cytosine residue (mSc), and rarely
at position 4 of a cytosine residue (mnc) (for a review, cf [128]).
Methylation renders the DNA resistant to cleavage at the modified site.
Methylation at other positions within the recognition sequence by other
methyltransferases, including dam, dcm, and eukaryotic methyl-
transferases, may or may not affect the rate of DNAcleavage by a restric-
tion enzyme. For example, DNA containing the site -AGGCCTGG- is
not cleaved by AatI or StuI when the DNA is extracted from a dcm ÷
strain orE. coIi, since in this case, the recognition site forAatI or StuI
overlaps with a dcm methylation site (129). Although in this case
isoschizomers exhibit the same sensitivity, others do not, e.g., HpalI is
sensitive to methylation within its recognition sequence -CCGG-,
whereas MspI is not (130), or only to a very minor degree (131). This
differential sensitivity of HpalI and MspI can be used to analyze the
methylation state of genes of higher eukaryotes, which depending on
the transcriptional activity are more or less methylated at-CG- sequences.
Likewise, the isoschizomers MboI, Sau3AI, DpnI, and DpnlI cleave
DNA within the dam methylase recognition site -GATC-, but differ with
respect to their sensitivity toward methylation: MboI and DpnlI cleave
only -GATC-, and Sau3AI recognizes both -GATC- and -Gm6ATC -,
whereas DpnI only cleaves -Gm6ATC - (132). The effects of site-specific
methylation on restriction as well as modification enzymes have been
reviewed (128) and will, therefore, not be discussed in more detail. It
should be emphasized, however, and this will be mentioned later (Section
3.7.), that the combined use of modification and restriction enzymes can
be utilized to narrow down the specificity of a restriction enzyme.
2.4.2. "Star"Activity
The specificity of restriction enzymes can be relaxed by subopti-
mum buffer conditions. This phenomenon, defined as "star" activity,
was first observed by Polisky et el. (133), who noticed that, under