Cutting and joining DNA molecules - Principles of Gene Manipulation - page 30

These DNA fragments can associate by hydrogen
bonding between overlapping 5

Recognition sequences

termini, or the frag-
ments can circularize by intramolecular reaction
(Fig. 3.2). For this reason the fragments are said to
have sticky or cohesive ends. In principle, DNA frag-
ments from diverse sources can be joined by means
of the cohesive ends and, as we shall see later, the
nicks in the molecules can be sealed to form an intact
artificially recombinant DNA molecule.
Not all type II enzymes cleave their target sites like
Eco RI. Some, such as Pst I (CTGCA/G), produce frag-
ments bearing 3

′

Most, but not all, type II restriction endonucleases
recognize and cleave DNA within particular sequences
of four to eight nucleotides which have a twofold
axis of rotational symmetry . Such sequences are often
referred to as palindromes because of their similarity
to words that read the same backwards as forwards.
For example, the restriction and modification enzymes
R. Eco RI and M. Eco RI recognize the sequence:

5 ′ -GA A T T C-3 ′

overhangs, while others, such as
Sma I (CCC/GGG), produce blunt or flush ends.
To date, over 10 000 microbes from around the
world have been screened for restriction enzymes.
From these, over 3000 enzymes have been found
representing approximately 200 different sequence
specificities. Some representative examples are shown
in Table 3.3. For a comprehensive database of
information on restriction endonucleases and their
associated methylases, including cleavage sites,
commercial availability and literature references,
the reader should consult the website maintained by
New England Biolabs (www.rebase.neb.com).
Occasionally enzymes with novel DNA sequence
specificities are found but most prove to have the
same specificity as enzymes already known. Restric-
tion enzymes with the same sequence specificity and
cut site are known as isoschizomers. Enzymes that
recognize the same sequence but cleave at different
points, for example Sma I (CCC/GGG) and Xma I
C/CCGGG), are sometimes known as neoschizomers .
Under extreme conditions, such as elevated pH
or low ionic strength, restriction endonucleases are

′

3 ′ -C T T AAG-5 ′

Axis of symmetry

The position at which the restricting enzyme cuts is
usually shown by the symbol '/' and the nucleotides
methylated by the modification enzyme are usually
marked with an asterisk. For Eco RI these would be
represented thus:

5 ′ -G/AA* T T C-3 ′

3 ′ -C T T A* A/G-5 ′

For convenience it is usual practice to simplify the
description of recognition sequences by showing
only one strand of DNA, that which runs in the 5 ′ to

3 direction. Thus the Eco RI recognition sequence

would be shown as G/AATTC.
From the information shown above we can see
that Eco RI makes single-stranded breaks four bases
apart in the opposite strands of its target sequence so
generating fragments with protruding 5

′

′ termini:

5 ′ -G 5 ′ -AATTC-3 ′

3 ′ -CTTAA-5 ′ G-5 ′

Intermolecular associatIon Intramolecular association

AATT AATT

5' 5'

5'

5'

TTAA

TTAA
Base - pairing

5' AATT

5'

AATT

A A T T

T T A A

Base - pairing

AATT AATT

5'

Fig. 3.2 Cohesive ends of DNA fragments produced
by digestion with Eco RI.

5'

TTAA TTAA