Restriction endonuclease enzymes occur naturally in bacteria as a chemical weapon against the invading viruses. They cut both strands of DNA when certain foreign nucleotides are introduced in the cell. Endonucleases break strands of DNA at internal positions in random manner.
Types of Restriction Enzymes:
1. Restriction enzyme Type I:
These enzymes interact with an unmodified recognition sequence in double-stranded DNA and then attach to long DNA molecule. After travelling for distance between 1000 to 5000 nucleotides the enzymes cleaves only one strand of the DNA at an apparently random site, and creates a gap of about 75 nucleotides in length.
Acid soluble oligonucleotides removed from the gap are released. A second enzyme molecule is needed to cleave the remaining strand of DNA. The cofactors for the enzyme are Mg2+ ions, ATP and S-adenosyl- methionine. This kind of enzyme is not useful for genetic engineering, because its cleavage sites are non-specific.
Type I restriction enzyme can simultaneously hold two different sites on DNA creating a loop in nucleic acid. This enzyme consists of three types of subunits. The Eco K enzyme, for example, has the structure R2M2S.
The R subunit is responsible for restriction and the M subunit for methylation. The binding of enzyme to DNA may be succeeded by either restriction or modification and this property is characterized by S subunit.
2. Restriction enzyme Type II:
These enzymes recognise a particular target sequence in a double-stranded DNA molecule. They cleave the polynucleotide chain within or near that sequence to give rise to distinct DNA fragments of defined length and sequence. They require Mg2+ ions for the action (i.e., restriction). Type II enzymes are used for gene manipulation studies.
3. Restriction enzyme Type III:
These enzymes cleave double-stranded DNA at well-defined sites. They require ATP, Mg2+ ions and have very partial requirement for S-adenosyl-methionine for restriction. They have intermediate properties between Type I and Type II REs.
Naming of Restriction Endonuclease Enzymes:
About 350 types of restriction endonucleases have been isolated from more than 200 bacterial strains. Large number of these enzymes require a system of uniform nomenclature. A system based on the proposals of Smith and Nathans (1973) has been followed for the most part.
Naming exercise of RE enzymes is based on following rules:
1. Each RE enzyme is named by a three-letter code.
2. The first letter of this code is derived from the first epithet (first letter of name) of the genus name. It is printed in italics.
3. The second and third letters are from the first two letters of its species name. They are also printed in italics.
4. This is followed by the strain number. If a particular strain has more than one restriction enzyme, these will be identified by Roman numerals as I, II, III, etc.
For example, the enzyme Eco RI was isolated from the bacterium Escherichia (E) coli (co) strain RY13 (R) and it was the first endonuclease (I). R also indicates antibiotic resistant plasmid of the bacterium. Likewise, Hind II from Haemophilus influenzae strain Rd and Bgl I from Bacillus globigii. A few restriction endonuclease enzymes and their sources are given in Table 55.3.
*Pu = purine
Target Sites of Restriction Endonuclease Enzymes:
A restriction endonuclease enzyme of type H recognises a specific recognition site (base sequence) on the DNA and makes a cut at this site only. These target sites are 4 to 6 nucleotides long (Fig. 55.3).
They exhibit palindromic symmetry, i.e., nucleotide pair sequences are same reading forward or backward from a central axis of symmetry, like the nonsense phrase-AND ‘MADAM DNA’.
The term palindromic has also been applied to sequences such as:
5′-AGCCGA—
3′-TCGGCT—
both of which are palindromic strands.
X-ray crystallography of RE enzyme-DNA complex indicate that endonuciease acts as a dimer of identical subunits and that the palindromic nature of target sequence reflects the two fold rotational symmetry of the dimeric protein.
Nature of Cut Ends:
Two types of cut ends of DNA, namely blunt or flush ends and sticky or cohesive ends, are produced by the restriction endonuclease s. The nature of these cut ends generated by the REs are very important in designing the gene cloning experiments.
1. Blunt cut ends:
In case of the blunt cut end, the enzyme (e.g., Haelll, Smal) makes a simple double-stranded cut in the middle of the recognition sequence. Thus the blunt ends or flush ends are formed. The RE Hae III makes a cut in the 5′-GGCC-3′ target site as shown in Fig. 55.3.
The utility of generation of blunt end cuts during the joining of DNA fragments is that any pair of ends may be joined together irrespective of sequence. This is especially useful for those researchers who are interested to join two defined sequences without introducing any additional material between them. Table 55.4 shows certain blunt-end restriction sites.
2. Sticky-or Cohesive ends:
Many restriction enzymes (e.g., Eco RI, Bam HI, and Hind III) make staggered, single-stranded cuts, producing short single-stranded projections at each end of the cleaved DNA, called sticky ends.
Since the restriction sites are symmetrical, so that both strands have the same sequence when read in the 5′ to 3′ direction. Thus, such staggered cuts will generate identical single-stranded projections on the either site of the cut (Fig. 55.4).
These ends are not only identical, but complementary, and will base pair with each other; they are therefore, known as cohesive or sticky ends. Because of specificity of restriction enzymes, every copy of a given DNA molecule will give the same set of fragments when cleaved with a particular enzyme.
Different DNA molecules will in general, give different sets of fragments when treated with the same enzyme. The table 55.5 shows sticky or cohesive-end restriction enzymes and sites:
Host Controlled Restriction and Modification:
Certain strains of bacteria are immune to bacteriophages. This phenomenon is called host controlled restriction. This restriction is due to these restriction endonuclease enzymes (e.g., Eco RI) which could recognise and split specific loci in the foreign DNA. Thus these enzymes prevent or restrict the survival of foreign DNA in the host. This is analogous to an immune system.
All restriction sites in host chromosome of a bacterium are protected from its own restriction endonuclease enzyme due to a modification system. This system helps in preventing suicidal self-degradation.
Such modification occurs by methylation of specific bases in the recognition sequence of the endonuclease. The enzymes involved in such modification are called methyltransferases.
These enzymes methylate adenine (i.e., adds a methyl group to the base) in the N6 position and cytosine either in N5 or W position and produce 6 methyl adenine and 5 or 4 methyl cytosine respectively.
Unmodified foreign DNA entering the cell is degraded by the host restriction system. As both the enzymes, i.e., methyltransferases and endonucleases recognise the restriction site, they are together called as restriction and modification system.
Star Activity:
Various REs show , the star activity when they exhibit relaxation in specificity of sequence under non-optimal conditions. In such condition, endonuclease enzymes even recognise other alternative base instead of a specific base.
The following factors are known to alter the DNA recognition sequence for several to alter the DNA recognition sequence for several endonuclease enzymes: non-ideal strength buffers, high glycerol concentration (more than 5% v\v) and high enzyme concentration.
Isoschizomers:
Isoschizomers are restriction endonuclease enzymes which are isolated from different organisms but recognize identical base sequences in the DNA. For example, Asp 718 and Kpn I have identical recognition sites-
Source of Asp718 is Achromobacter species 718; source of Kpn I is Klebsiella pneumoniae OK 8. Some pairs of isochizomers cut their target at different places (e.g., Sma I, Xma I).
Use of Restriction Endonuclease Enzymes in Genetic Engineering:
In gene cloning experiments, DNA molecules have to be cut in a very precise and reproducible manner. Restriction endonuclease enzymes play an important role in cutting the desired gene as well as cleaving the vector.
1. Cutting the gene:
The required DNA fragment from a large DNA molecule should be cleaved in a precise manner for further genetic manipulations. A particular restriction endonuclease enzyme can recognize and bind to specific base sequence of the DNA and then will cleave it. It is highly reproducible and can be programmed according to DNA sequences of required gene and particular endonuclease enzymes identifying and cleaving it.
2. Cutting the vectors:
The function of a vector DNA molecule is to carry a gene of interest to a second organism where it can express it (i.e., can produce a gene specific product). During this technique the DNA to be cloned is integrated with the plasmid.
Hence each vector molecule should be cleaved with same restriction site at a single position to open the circular form so that the new DNA fragment can be inserted at these complementary sites.
If foreign DNA is introduced into E. coli host, it may be attacked by restriction endonucleases active in a host cell. Because restriction phenomenon 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 DNA molecules. This will eliminate the chance that the incoming sequence will be restricted.