What is a RESTRICTION ENZYME?

A restriction enzyme is a protein that recognizes

a specific, short nucleotide sequence and cuts the
DNA only at that specific site, which is known
as restriction site or target sequence.
What is the function of a restriction endonuclease?
In cells restriction endonucleases are used to
cut up invading DNA from viruses.
The restriction sites are ones found in viral DNA.
If, when a virus injects DNA in a bacterium,
the restriction enzymes cut up the viral DNA then
it cannot take over the bacterium and cause it to
manufacture more viruses.

How do restriction enzymes cleave DNA?

A major protective strategy for the host is to
use restriction endonucleases(restriction
enzymes) to degrade the viral DNA on its
introduction into a cell. These enzymes recognize
particular base sequences, called recognition
sequences or recognition sites, in their
target DNA and cleave that DNA at defined
positions.

Types of Restriction Enzymes:

Restriction enzymes are classified biochemically
into three types. These are designated as Type I,
Type II, and Type III. A major type of Type II
enzymes are sometimes referred to as Type IV
enzymes.
Type I and III systems, both the methylase and
restriction activities are carried out by a single
large enzyme complex. Although these enzymes
recognize specific DNA sequences, the sites of
actual cleavage are at variable distances from
these recognition sites, and can be hundreds of
bases away. Both require ATP for their proper
function.
Type I restriction enzymes produce DNA cleavage
following translocation of the DNA, which makes
them important molecular motors. The cleavage of
DNA appears to occur after blockage of the
translocation activity (often following collision with
another translocating Type R-M enzyme, but also
due to other factors).
These enzymes read the methylation status of their
recognition sequence, compare the methylation
status of two adenines within the recognition
sequence, and if both adenines are un-methylated
(a signal that the DNA is non-host DNA), the
enzyme undergoes a conformational switch that

turns the enzyme into a molecular motor and

endonuclease.
However, if either one of the adenines is
methylated (a signal that the DNA is host DNA)
then the enzyme acts as a maintenance methylase
and methylates the other adenine. In Type II
systems, the restriction enzyme is independent of
its methylase, and cleavage occurs at very specific
sites that are within or close to the recognition
sequence. The vast majority of known restriction
enzymes are of type II, and it is these that find the
most use as laboratory tools. They produce
discrete bands during gel electrophoresis, and are
useful for DNA analysis and gene cloning. The first
to be discovered and utilized was EcoRI, which is
staggered and its recognition sequence is 5GAATTC-3.
Type II enzymes are further classified according to
their recognition site. Most type II enzymes cut
palindromic DNA sequences, while type IIa
enzymes recognise non-palindromic sequences and
cleave outside of the recognition site, and type lIb
enzymes cut sequences twice at both sites outside
the recognition sequence. Type lIs enzymes cleave
the DNA at a considerable offset from the
recognition sequence. The most common type II
enzymes are those like HhaI, HindIII and NotI that
cleave DNA within their recognition sequences.

Restriction enzymes usually occur in combination

with one or two modification enzymes (DNAmethyltransferases) that protect the cells own
DNA from cleavage by the restriction enzyme.
Modification enzymes recognize the same DNA
sequence as the restriction enzyme that they
accompany, but instead of cleaving the sequence,
they methylate one of the bases in each of the
DNA strands. The methyl groups protrude into the
major groove of DNA at the binding site and
prevent the restriction enzyme from acting upon it.
Together, a restriction enzyme and its cognate
modification enzyme(s) form a restrictionmodification (R-M) system. In some R-M systems
the restriction enzyme and the modification
enzyme(s) are separate proteins that act
independently of each other. In other systems, the
two activities occur as separate subunits, or as
separate domains, of a larger, combined,
restriction-and-modification enzyme.

Restriction Enzymes as Tools:

Recognition sequences typically are only four to
twelve nucleotides long. Because there are only so
many ways to arrange the four nucleotidesA,C,G

and Tinto a four or eight or twelve nucleotide

sequence, recognition sequences tend to crop up
by chance in any long sequence. Furthermore,
restriction enzymes specific to hundreds of distinct
sequences have been identified and synthesized
for sale to laboratories.
As a result, potential restriction sites appear in
almost any gene owr chromosome. Meanwhile, the
sequences of some artificial plasmids include a
linker that contains dozens of restriction enzyme
recognition sequences within a very short segment
of DNA. So no matter the context in which a gene
naturally appears, there is probably a pair of
restriction enzymes that can cut it out, and which
will produce ends that enable the gene to be
spliced into a plasmid.
Another use of restriction enzymes can be to find
specific SNPs. If a restriction enzyme can be found
such that it cuts only one possible allele of a
section of DNA (that is, the alternate nucleotide of
the SNP causes the restriction site to no longer
exist within the section of DNA), this restriction
enzyme can be used to genotype the sample
without completely sequencing it. The sample is
first run in a restriction digest to cut the DNA, and
then gel electrophoresis is performed on this
digest.

If the sample is homozygous for the common allele,

the result will be two bands of DNA, because the
cut will have occurred at the restriction site. If the
sample is homozygous for the rarer allele, the
sample will show only one band, because it will not
have been cut. If the sample is heterozygous at
that SNP, there will be three bands of DNA. This is
an example of restriction mapping.

How is restriction enzymes used in recombinant

DNA technology?
MESSAGE. Restriction enzymes have two
properties useful in recombinant DNA
technology. First, they cut DNA into fragments of
a size suitable for cloning. Second,
many restriction enzymes make staggered cuts
that create single-stranded sticky ends conducive
to the formation of recombinant DNA.

How many restriction enzymes are there?

More than 3000 type II restriction endonucleases
have been discovered. They recognize short,
usually palindromic, sequences of 48 bp and, in

the presence of Mg2+, cleave the DNA within or in

close proximity to the recognition sequence. The
orthodox type II enzymes are homodimers which
recognize palindromic sites.
More than 400 restriction enzymes have been
isolated from the bacteria that manufacture them.
In live bacteria, restriction enzymes function to
defend the cell against invading viral
bacteriophages. Restrictions sites in the viral
genome (a "happy accident" of nature, as far as
the bacteria are concerned, since they don't
appear to have any specific function in the virus)
are cleaved by the bacterium's restriction
enzymes, fragmenting and destroying the DNA of
invading bacteriophages before it can incorporate
into the host's genome and take over the cell.
A bacterium is immune to its own restriction
enzymes, even if it has the target sequences
ordinarily targeted by them. This is because the
bacterial restriction sites are highly methylated,
making them unrecognizable to the restriction
enzyme.
Restriction enzymes are named based on the organism in which they were
discovered. For example, the enzyme Hind III was isolated from Haemophilus
influenzae, strain Rd. The first three letters of the name are italicized because
they abbreviate the genus and species names of the organism. The fourth letter
typically comes from the bacterial strain designation. The Roman numerals
are used to identify specific enzymes from bacteria that contain multiple
restriction enzymes. Typically, the Roman numeral indicates the order in

which restriction enzymes were discovered in a particular strain.

Read more: Restriction Enzymes - Nomenclature And Classification - Dna,
Type, Recognition, and Sequences - JRank
Articles http://medicine.jrank.org/pages/2778/Restriction-EnzymesNomenclature-Classification.html#ixzz4UhSZewQI

Patterns of DNA Cutting by Restriction Enzymes:

(i) 5 overhangs:
ADVERTISEMENTS:

The enzyme cuts asymmetrically within the recognition site such that a
short single-stranded segment extends from the 5 ends. BamHI cuts
in this manner.

(ii) 3 overhangs:
Again, we see asymmetrical cutting within the recognition site, but the
result is a single-stranded overhang from the two 3 ends. Kpnl cuts in
this manner.

(iii) Blunts:
Enzymes that cut at precisely opposite sites in the two strands of DNA
generate blunt ends without overhangs. Smal is an example of an
enzyme that generates blunt ends.

The 5 or 3 overhangs generated by enzymes that cut asymmetrically

are called sticky ends or cohesive ends, because they will readily stick
or anneal with their partner by base pairing.

here are three main groups of restriction endonucleases (REases) called Types I, II, and III (1,2). Since
1973, REases and DNA methyltransferases (MTases) have been named based on an original suggestion
by Smith and Nathans (3). They proposed that the enzyme names should begin with a three-letter
acronym in which the first letter was the first letter of the genus from which the enzyme was isolated and
the next two letters were the first two letters of the species name. Extra letters or numbers could be added
to indicate individual strains or serotypes. Thus, the enzyme Hindll was one of four enzymes isolated
from H aemophilus in fluenzae serotype d.The first three letters of the name were italicized. Later, a
formal proposition for naming the genes encoding REases and MTases was adopted (4). When there
were only a handful of enzymes known, these schemes were very useful, but as more enzymes have
been found, often from different genera and species with names whose three-letter acronyms would be
identical, considerable laxity in naming conventions has appeared. In addition, we now know that each
major type of enzyme can contain subtypes. This especially applies to the Type II enzymes, of which
more than 3500 have been characterized (5). In this paper we revisit the naming conventions and outline
an updated scheme that incorporates current knowledge about the complexities of these enzymes. We
describe a set of naming conventions for REases and their associated MTases.