RESTRICTION ENZYMES
A restriction enzyme (or restriction endonuclease)
is an enzyme that cuts
double-stranded DNA following
its specific recognition of short nucleotide sequences, known as restriction
sites, in the DNA. They are found in bacteria and archaea. Restriction
enzymes selectively cut up foreign
DNA in a process called restriction.
Host DNA is methylated
by a modification enzyme (a methylase) to protect it from the restriction enzyme’s
activity. Collectively, these two processes form the Restriction
Modification System. A restriction enzyme makes two incisions,
once through each sugar-phosphate backbone (i.e. each strand) of the DNA double
helix. Daniel Nathans, Werner Arber, and Hamilton
Smith (Nobel Prize
in Medicine in 1978) were awarded with Nobel Prize for the discovery of
restriction endonucleases. They are routinely used for DNA modification and
manipulation in laboratories.
Contents
4. Mechanism for RE
1.
Recognition sites
5'-GAATTC-3'
||||||||||||||||||
3'-CTTAAG-5'
||||||||||||||||||
3'-CTTAAG-5'
Figure 1: Screening of Recognition site by EcoRI
A palidromic recognition site reads the same
on the reverse strand as it does on the forward strand. Restriction
enzymes recognize a specific sequence of nucleotides, the lengths vary between
4 and 8 nucleotides, many of them are palindromics. The meaning of "palindromic" in this
context is different from what one might expect from its linguistic usage:
GTAATG is not a palindromic DNA sequence, but GTATAC is (GTATAC is complementary
to CATATG). E.g. EcoRI digestion produces "sticky" ends while 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 the 3' end)
of a sticky-end "overhang"
of an enzyme restriction. Bacteria prevent their own DNA from being cut by
modifying their nucleotides via methylation.
Figure 2: Sticky ends generated after restriction digestion
2. Enzyme classes
There are three general 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.
Type I
It is first to be identified. It is a characteristic of two
different strains (K-12 and B) of E. coli.
It cuts at a site that differs, and is some distance away, from their
recognition site. It has asymmetrical recognition site and is composed of two
portions – one containing 3-4 nucleotides, and another containing 4-5
nucleotides – separated by a spacer of about 6-8 nucleotides. Several enzyme
cofactors, including S-Adenosyl methionine (AdoMet), hydrolyzed
adenosine triphosphate (ATP)
and magnesium (Mg2+)
ions, are required for
their activity. They possess three subunits called HsdR, HsdM, and HsdS (HsdR is required for restriction, HsdM is
necessary for adding methyl
groups to host DNA (methyltransferase activity) and HsdS is important for
specificity of cut site recognition in addition to its methyltransferase
activity).
(The hsd genes of E
coli K12 have been cloned in phage lambda by a combination of in vitro and in
vivo techniques. Three genes, whose products are required for K-specific
restriction and modification, have been identified by complementation tests as
hsdR, hsdM and hsdS. The order of these closely linked genes was established as
R, M and S by analysis of the DNA of genetically characterized deletion
derivatives of lambda hsd phages. The three genes are transcribed in same
direction but not necessarily as a single operon. Genetic evidence identifies two
promoters, one from which transcription of hsdM and S is initiated and a second
for the hsdR gene. The hsdR gene codes for a polypeptide of Mw approx. 130000;
hsdM for one 62-65000 and the hsdS gene was associated with polypeptide of
approx 50000. Circumstantial evidence suggests that one of these two
polypeptide may be degradation, or processed derivative of the other. The hsdS
polypeptide of e coli B has a slightly higher mobility in an SDS-PAGE than does
that of E coli K12. A probe comprising most of the hsdR gene and all of the
hsdM and S genes of Ecoli k12 shares extensive homology with the DNA of EcoliB
but none with that of Ec oil C.)
Type II
Typical type II restriction enzymes
differ from type I restriction enzymes in several ways. They are composed of
only one subunit. Recognition sites are usually undivided and palindromic and 4-8
nucleotides in length. They recognize and cleave DNA at the same site. They do
not use ATP or AdoMet for their activity and usually require only Mg2+
as a cofactor. They are the most commonly available and used restriction
enzymes.
Type IIB restriction enzymes (e.g. BcgI and BplI)
are multimers, containing more
than one subunit. They cleave DNA on both sides of their recognition to cut out
the recognition site. They require both AdoMet and Mg2+ cofactors.
Type IIE restriction endonucleases (e.g. NaeI) cleave DNA following
interaction with two copies of their recognition sequence.[13]
One recognition site acts as the target for cleavage, while the other acts as
an allosteric
effector that speeds up or improves the efficiency of enzyme
cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases (e.g.
NgoMIV) interact with two copies of their recognition sequence but
cleave both sequences at the same time. Type IIG restriction endonucleases (Eco57I)
do have a single subunit, like classical Type II restriction enzymes, but
require the cofactor AdoMet to be active.[13]
Type IIM restriction endonucleases, such as DpnI, are able to recognize
and cut methylated DNA. Type IIS restriction endonucleases (e.g. FokI)
cleave DNA at a defined distance from their non-palindromic asymmetric
recognition sites. These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g. Bpu10I
and BslI) are composed of two different subunits. Some recognize
palidromic sequences while others have asymmetric recognition sites.
Type
III
They 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.
3.
Nomenclature
Since their discovery in the 1970s, more than 100 different
restriction enzymes have been identified in different bacteria. 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.
E
|
Escherichia
|
(genus)
|
co
|
coli
|
(species)
|
R
|
RY13
|
(strain)
|
I
|
First identified
|
(order of identification in the
bacterium)
|
4.
Mechanism of Restriction Endonuclease:
Isoschizomers and Neoschizomers:
Restriction enzymes that
have the same recognition sequence as well as the same cleavage site are
Isoschizomers.
Restriction enzymes that
have the same recognition sequence but cleave the DNA at a different site
within that sequence are Neoschizomers. Eg: SmaI and XmaI
C C
C G G G C C C G G G
G G
G C C C G
G G C C C
Xma
I Sma
I
Restriction
Endonuclease scan the length of the DNA and binds to the DNA molecule when it
recognizes a specific sequence and makes one cut in each of the sugar phosphate
backbones of the double helix – by hydrolyzing the phoshphodiester bond.
Specifically, the bond between the 3’ O atom and the P atom is broken as shown
in figure below.
Figure 3: Break down of phosphodiester bonding in DNA substrate
Then,
3’OH and 5’ PO43- is produced. Mg2+ is
required for the catalytic activity of the enzyme. It holds the water molecule
in a position where it can attack the phosphoryl group and also helps polarize
the water molecule towards deprotonation.
The
enzyme consists of two subunits –dimers related by two fold rotational
symmetry. It binds to the matching symmetry of the DNA molecule at the
restriction site and produces a kink at the site.
Figure 4: (A) 3D structure of endonuclease, EcoRv (B) Binding of DNA to endonuclease (C) Bresk down of hydrogen bonding between N-bases
5.
Restriction enzymes as tools
- They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments.
- They can be used to distinguish gene alleles (member of a pair or series of different forms of a gene) by specifically recognizing single base changes in the DNA known as single nucleotide polymorphisms (SNPs). This is only possible if a restriction site, present in one allele is altered by the SNP in the second allele.
- In a similar manner, restriction enzymes are used to digest genomic DNA for gene analysis by Southern Blot.
Figure 5: Restriction map analysis and its application in Southern hybridization
6. Examples
Tabel 2: Examples of restriction endonucleases with their recognition sites
Enzyme
|
Source
|
Recognition Sequence
|
Cut
|
5'GAATTC
3'CTTAAG
|
5'---G AATTC---3'
3'---CTTAA G---5'
|
||
5'CCWGG
3'GGWCC
|
5'--- CCWGG---3'
3'---GGWCC ---5'
|
||
5'GGATCC
3'CCTAGG
|
5'---G GATCC---3'
3'---CCTAG G---5'
|
||
5'AAGCTT
3'TTCGAA
|
5'---A AGCTT---3'
3'---TTCGA A---5'
|
||
5'TCGA
3'AGCT
|
5'---T CGA---3'
3'---AGC T---5'
|
||
5'GCGGCCGC
3'CGCCGGCG
|
5'---GC GGCCGC---3'
3'---CGCCGG CG---5'
|
||
5'GANTC
3'CTNAG
|
5'---G ANTC---3'
3'---CTNA G---5'
|
||
5'GATC
3'CTAG
|
5'--- GATC---3'
3'---CTAG ---3'
|
||
5'CAGCTG
3'GTCGAC
|
5'---CAG CTG---3'
3'---GTC GAC---5'
|
||
5'CCCGGG
3'GGGCCC
|
5'---CCC GGG---3'
3'---GGG CCC---5'
|
||
5'GGCC
3'CCGG
|
5'---GG CC---3'
3'---CC GG---5'
|
||
5'AGCT
3'TCGA
|
5'---AG CT---3'
3'---TC GA---5'
|
||
5'GATATC
3'CTATAG
|
5'---GAT ATC---3'
3'---CTA TAG---5'
|
||
5'GGTACC
3'CCATGG
|
5'---GGTAC C---3'
3'---C CATGG---5'
|
||
5'CTGCAG
3'GACGTC
|
5'---CTGCA G---3'
3'---G ACGTC---5'
|
||
5'GAGCTC
3'CTCGAG
|
5'---GAGCT C---3'
3'---C TCGAG---5'
|
||
5'GTCGAC
3'CAGCTG
|
5'---G TCGAC---3'
3'---CAGCT G---5'
|
||
5'AGTACT
3'TCATGA
|
5'---AGT ACT---3'
3'---TCA TGA---5'
|
||
5'GCATGC
3'CGTACG
|
5'---G CATGC---3'
3'---CGTAC G---5'
|
||
5'AGGCCT
3'TCCGGA
|
5'---AGG CCT---3'
3'---TCC GGA---5'
|
||
5'TCTAGA
3'AGATCT
|
5'---T CTAGA---3'
3'---AGATC T---5'
|
||
* = blunt ends
|
|||
N = C or G or T or A
|
|||
W = A or T
|
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