Restriction Enzymes:
A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into
fragments at or near specific recognition sites within molecules known as restriction sites. Restriction
enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are
commonly classified into five types, which differ in their structure and whether they cut their DNA
substrate at their recognition site, or if the recognition and cleavage sites are separate from one another.
To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone
(i.e. each strand) of the DNA double helix.
These enzymes are found in bacteria and archaea and provide a defence mechanism against invading
viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called
restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase)
that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the
restriction modification system.
Over 3,000 restriction enzymes have been studied in detail, and more than 600 of these are available
commercially. These enzymes are routinely used for DNA modification in laboratories, and they are a
vital tool in molecular cloning.
Fig 1: Restriction modification system of a bacterial species
Recognition site
A palindromic recognition site reads the same on the reverse strand as it does on the forward strand
when both are read in the same orientation.
Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in
the DNA. The recognition sequences can also be classified by the number of bases in its recognition site,
usually between 4 and 8 bases, and the number of bases in the sequence will determine how often the
site will appear by chance in any given genome, e.g., a 4-base pair sequence would theoretically occur
once every 4^4 or 256bp, 6 bases, 4^6 or 4,096bp, and 8 bases would be 4^8 or 65,536bp. Many of them
are palindromic, meaning the base sequence reads the same backwards and forwards. In theory, there
are two types of palindromic sequences that can be possible in DNA. The mirror-like palindrome is
similar to those found in ordinary text, in which a sequence reads the same forward and backward on a
single strand of DNA, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the
same forward and backward, but the forward and backward sequences are found in complementary DNA
strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG).
Inverted repeat palindromes are more common and have greater biological importance than mirror-like
palindromes.
Fig 2: Types of palindromes
Types of cut by the restriction Enzymes:
EcoRI digestion produces "sticky" ends,
whereas SmaI restriction enzyme cleavage produces "blunt" ends:
Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length,
sequence and strand orientation (5' end or 3' end) of a sticky-end "overhang" of an enzyme restriction.
Different restriction enzymes that recognize the same sequence are known as neoschizomers. These
often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same
location are known as isoschizomers.
Types of Restriction Enzymes:
Naturally occurring restriction endonucleases are categorized into three groups (Types I, II and III) based
on their composition and enzyme cofactor requirements, the nature of their target sequence, and the
position of their DNA cleavage site relative to the target sequence. DNA sequence analyses of restriction
enzymes however show great variations, indicating that there are more than four types. All types of
enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to
give specific fragments with terminal 5'-phosphates. They differ in their recognition sequence, subunit
composition, cleavage position, and cofactor requirements, as summarised below:
Type I enzymes cleave at sites remote from a recognition site; require both ATP and S-adenosyl-L-
methionine to function; multifunctional protein with both restriction digestion and methylase
activities.
Type II enzymes cleave within or at short specific distances from a recognition site; most require
magnesium; single function (restriction digestion) enzymes independent of methylase.
Type III enzymes cleave at sites a short distance from a recognition site; require ATP (but do not
hydrolyse it); S-adenosyl-L-methionine stimulates the reaction but is not required; exist as part of
a complex with a modification methylase
Type I
Type I restriction enzymes were the first to be identified and were first identified in two different strains
(K-12 and B) of E. coli. These enzymes cut at a site that differs, and is a random distance (at least 1000
bp) away, from their recognition site. These enzymes are multifunctional and are capable of both
restriction digestion and modification activities, depending upon the methylation status of the target
DNA. The cofactors S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and
magnesium (Mg2+) ions, are required for their full activity. Type I restriction enzymes possess three
subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction digestion; HsdM is necessary for
adding methyl groups to host DNA (methyltransferase activity), and HsdS is important for specificity of
the recognition (DNA-binding) site in addition to both restriction digestion (DNA cleavage) and
modification (DNA methyltransferase) activity.
Type II
Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They form
homodimers, with recognition sites that are usually undivided and palindromic and 4–8 nucleotides in
length. They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their
activity—they usually require only Mg2+ as a cofactor. These enzymes cleave the phosphodiester bond of
double helix DNA. It can either cleave at the center of both strands to yield a blunt end, or at a staggered
position leaving overhangs called sticky ends. These are the most commonly available and used
restriction enzymes.
Type III
Type III restriction enzymes (e.g., EcoP15) recognize two separate non-palindromic sequences that are
inversely oriented. They cut DNA about 20–30 base pairs after the recognition site. These enzymes
contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation
and restriction digestion , respectively. They are components of prokaryotic DNA restriction-modification
mechanisms that protect the organism against invading foreign DNA. Type III enzymes are hetero-
oligomeric, multifunctional proteins composed of two subunits, Res and Mod. The Mod subunit
recognises the DNA sequence specific for the system and is a modification methyltransferase; as such, it is
functionally equivalent to the M and S subunits of type I restriction endonuclease. Res is required for
restriction digestion, although it has no enzymatic activity on its own.
Fig 3: Properties of restriction enzymes
Nomenclature:
Derivation of the EcoRI name
Abbreviation Meaning Description
E Escherichia genus
co coli specific species
R RY13 strain
order of identification
I First identified
in the bacterium
Since their discovery in the 1970s, many restriction enzymes have been identified; for example, more
than 3500 different Type II restriction enzymes have been characterized. Each enzyme is named after the
bacterium from which it was isolated, using a naming system based on bacterial genus, species and strain.
For example, the name of the EcoRI restriction enzyme was derived as shown in the box.
Examples:
Examples of restriction enzymes include:
Fig 4: Examples of Restriction Enzymes
Applications of Restriction Enzymes:
Isolated restriction enzymes are used to manipulate DNA for different scientific applications.
They are used to assist insertion of genes into plasmid vectors during gene cloning and protein
production experiments. To clone a gene fragment into a vector, both plasmid DNA and gene insert are
typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme
known as a DNA ligase. In this case, insecticide gene is inserted into the plasmid vector. Growing plant
cells take up the insecticide gene from the plasmid vector. Cells propagate into new plants. Since these
plants consist of insecticides, any insect trying to feed on these plants will die.
