Nomenclature:
The discovery of a large number of restriction enzymes called for a uniform nomenclature. A system based upon the proposals of Smith & Nathans (1973) has been followed for ihc most part. The proposals were as follows:
(I) The species name of the host organism is identified by the first letter of the genus name and the first two letters of the specific epithet to form a three-letter abbreviation in italics. For example, Escherichia coil = Eco and Hoemophilus irtfluenzsie — Hin.
(2) Strain or type identification is written as a subscript, e.g. Eco. In cases where the restriction and modification system is genetically specified by a virus or plasmid. the abbreviated species name of the host is given and the cxtrachromosomal element is identified by a subscript, e.g. EcoN. Eco1.
(3) When a particular host strain has several different restriction and modification systems, these are identified by Roman numerals, thus the systems from H. in.fluenae strain Rd would be Hsndl. Hlnll. Hind111.
(4) All restriction enzymes have the general name endonucleasc R. but, in addition, carry the system name, e.g. endonuclease R. Hindu1. Similarly, modification enzymes arc named met hylasc M followed by the system name. The modification enzyme from H. Influenzoe Rd corresponding to cndonuclcase R. HindlIl is designated mcthybsc M. Hindlll.
In practice this system of nomenclature has been simplified further.
(I) Subscripts are typographically inconvenient: the whole abbreviation is now usually written on the line.
(2) Where the context makes it clear that restriction enzymes only are involved, the designation endonucleasc R. is omitted. This is the system
used in Table 2.1 • which lists some of the more commonly used restriction cndonucleases.
Type II restriction endonucleases recognize and break DNA within particular sequences of ictra- , pcnta• hexa- or heptanucicotides which have an axts of rotational symmetry. For example. Eco RI cuts at the
There are also several known examples of enzymes from different sources which recognize the same target. They are isoschi:ome,s. Some pairs of isoschizomcrs cut their target at different places (e.g Sma I, Xma I).
Recently, a third class of rcsirction cndonucleasc has been idcniilicd. Type Ill enzymes make breaks in the Iwo strands at measured d&anct’s to one side of their target sequence (e.g. Hga 1). This variety of propenies exhibited by restriction endonucleases provides scope for the ingenious and resourceful gcnc manipubwr. What is the function of restriction endonuclcases in vivo? Clearly hostc onirolkd restriction acts as a mechanism by which bacteria distinguish self from non-self. It is analogous to an immunity system. Restnction is moderately effective in preventing infection by some bacteriophages. It may be for this reason that the T-even phages (T2. T4 and T6) have evolved with glucosylated hydroxymethylcytosine residues replacing cytosàne in their DNA. so rendering it resistant to many restriction endonucleases. The restriction and glucosylasion modification of T.cvcn phage DNA is beyond the scope of this book. For a detailed discussion the reader is referred to Kornbcrg (1974). However, it is worth noting that a mutant strain of T4 is available which does have cytosanc residues in its DNA and is therefore amenable to conventional restriction methodology (Veltcn er al. 1976). As an alternative to the unusual DNA structure of the T-cven phages,
othcr mcchanisms appcar to have cvolvcd in T3 and V for overcoming restriction in vivo (Spoerel et al. 1979). In spite of this evidence we may be mistaken in concluding that immunity (0 phage infection is the sole or main function of restriction endonucleascs in nature; loss or alteration of phagc receptors might be a more economical way of achieving immunity. For the present we can only speculate.
The discovery of a large number of restriction enzymes called for a uniform nomenclature. A system based upon the proposals of Smith & Nathans (1973) has been followed for ihc most part. The proposals were as follows:
(I) The species name of the host organism is identified by the first letter of the genus name and the first two letters of the specific epithet to form a three-letter abbreviation in italics. For example, Escherichia coil = Eco and Hoemophilus irtfluenzsie — Hin.
(2) Strain or type identification is written as a subscript, e.g. Eco. In cases where the restriction and modification system is genetically specified by a virus or plasmid. the abbreviated species name of the host is given and the cxtrachromosomal element is identified by a subscript, e.g. EcoN. Eco1.
(3) When a particular host strain has several different restriction and modification systems, these are identified by Roman numerals, thus the systems from H. in.fluenae strain Rd would be Hsndl. Hlnll. Hind111.
(4) All restriction enzymes have the general name endonucleasc R. but, in addition, carry the system name, e.g. endonuclease R. Hindu1. Similarly, modification enzymes arc named met hylasc M followed by the system name. The modification enzyme from H. Influenzoe Rd corresponding to cndonuclcase R. HindlIl is designated mcthybsc M. Hindlll.
In practice this system of nomenclature has been simplified further.
(I) Subscripts are typographically inconvenient: the whole abbreviation is now usually written on the line.
(2) Where the context makes it clear that restriction enzymes only are involved, the designation endonucleasc R. is omitted. This is the system
used in Table 2.1 • which lists some of the more commonly used restriction cndonucleases.
Type II restriction endonucleases recognize and break DNA within particular sequences of ictra- , pcnta• hexa- or heptanucicotides which have an axts of rotational symmetry. For example. Eco RI cuts at the
Sou,cc: Roberts (1971 . Rcc.rnton sequences arc written front S — only one strand being psen. and the potni o( ekasage Is indxatd by an anon. Bacs written rn p.icn4hcscs signify lisa. aiher base may occupy
that position. Where b.o.n. the base modified by the cortesponding\ specific methylase is indicated by an asterisk. A is N’.methyladcnine. C is S-mcrhyLcytossnc.
1.2. The names of these mo enzymes arc anomalous. The genes stifyitig the cnrnies are bat-ne on two Remàsgancc Tramler Factoes whidibasebccndasufiedwp.istcly. IlenceRland Ru,
3. Iig.l is a Ipe III rewntlon endonuclease. ckatng as indicated;
S’ GACGCNNNNNt
3 CTCICGNNNNNNNNNNI
4. Under certain condiuo.s (low ionic strength. .aikalinc pH 01 50!. g)ycuol) the Eco RI specificity is reduced so that only the internal ictranucleoticje sequence of the canonical bewwcleotide Is ncessazy
lo, recognition and cleavage. This Is so-called Ecu R1 (Rl.sW)actisily. It is inlubded by p.r.chloromcrcorlbenzoate. uhereas Ecu RI actny insitrse (T*lchonenko eviL 1978).
positions indicated by arrows in the sequence
axis of symmetry
I • I
5’—GAA TTC—
3’—CTT AAG—
giving rise to termini bearing 5 ‘-phosphate and 3 ‘-hydroxyl groups. Such
sequences are sometimes said to be palindromic by analogy with words
that read alike backwards and forwards. (However, (his term has also been
applied to sequences such as
5’ —AGCCGA—
3’ —TCGGCT--w
hich arc palindromic within one strand, yet do not have an axis of rotational symmetry.) If the sequence is modified by methylation so that
6-methyladenine residues are found at one or both of the positions indicated by asterisks then the sequence is resistant o cndonuclcase R. Eco RI. The resistance of the haif-methylated site protects the bacterial host’s own duplex DNA from attack immediately after semi-conservative replication of the fully-mcthylatcd site until the modification methylase can once again restore thc daughter duplcxes to the fully-methylatcd slate.
We can see that Eco RI makes single-strand breaks four nucleotide pairs apart in the opposite strands of its target sequence, and so generates fragments with protruding S ‘-termini. These DNA fragments can associate by hydrogen bonding between overlapping 5’ -tvrmini, or the fragments can circularize by intramolecular reaction, and for this reason the fragments are said to have sticky or cohesive ends (Fig. 2.2). In principle, DNA fragments from divcrsc sources can be joined by means of the cohesive ends, and n is possible, as we shall sec later, to seal the remaining nicks in the two strands to form an intact artificWIIy recombinant duplex DNAmolecule.
It is clear from Table 2.1 that not all type II enzymes have target sites like Eco RI. Some enzymes (e.g. P511) produce fragments bearing 3 ‘.cohesivc ends. Others (e.g. Hue Ill) make even cuts giving rise to flush• or bIunt ended fragmens with no cohesive end at all. Some enzymes recognize tetranudcotidc sequences, others recognize longer sequences. We would expect any particular tetranucleotide target to occur about once every 44 (i.e. 256) nucleotide pairs in a long random DNA sequence, assuming all bases are equally frequent. Any particular heanuclcotidc target would be expected to occur once in every 4’ (i.e. 4096) nudeotide pairs. Some enzymes (e.g. Mbo I) recognize a tctranuckotide sequence that is included within
the heanucleoiide sequence recognized by a different enzyme (e.g. Barn HI). Hand II, the first type II enzyme to be discovered, is an example of an enzyme recognizing a sequence with some ambiguity; in this case all three sequences corresponding to the structure given in Table 2.1 are substrates.
There are also several known examples of enzymes from different sources which recognize the same target. They are isoschi:ome,s. Some pairs of isoschizomcrs cut their target at different places (e.g Sma I, Xma I).
Recently, a third class of rcsirction cndonucleasc has been idcniilicd. Type Ill enzymes make breaks in the Iwo strands at measured d&anct’s to one side of their target sequence (e.g. Hga 1). This variety of propenies exhibited by restriction endonucleases provides scope for the ingenious and resourceful gcnc manipubwr. What is the function of restriction endonuclcases in vivo? Clearly hostc onirolkd restriction acts as a mechanism by which bacteria distinguish self from non-self. It is analogous to an immunity system. Restnction is moderately effective in preventing infection by some bacteriophages. It may be for this reason that the T-even phages (T2. T4 and T6) have evolved with glucosylated hydroxymethylcytosine residues replacing cytosàne in their DNA. so rendering it resistant to many restriction endonucleases. The restriction and glucosylasion modification of T.cvcn phage DNA is beyond the scope of this book. For a detailed discussion the reader is referred to Kornbcrg (1974). However, it is worth noting that a mutant strain of T4 is available which does have cytosanc residues in its DNA and is therefore amenable to conventional restriction methodology (Veltcn er al. 1976). As an alternative to the unusual DNA structure of the T-cven phages,
othcr mcchanisms appcar to have cvolvcd in T3 and V for overcoming restriction in vivo (Spoerel et al. 1979). In spite of this evidence we may be mistaken in concluding that immunity (0 phage infection is the sole or main function of restriction endonucleascs in nature; loss or alteration of phagc receptors might be a more economical way of achieving immunity. For the present we can only speculate.
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