RESTRICTION ENDONUCLEASES

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

1. Recognition sites

2. Enzyme classes

2.1. Type I

2.2. Type II

2.3. Type III

3. Nomenclatures

4. Mechanism for RE

5. Restriction enzymes as tools

6. Examples

 

1. Recognition sites

5'-GAATTC-3'
     |||||||||||||||||| 
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.

 Table 1: Nomencalture of restriction endonucleases

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’ PO₄^3- 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
EcoRI	Escherichia coli	5'GAATTC 3'CTTAAG	5'---G     AATTC---3' 3'---CTTAA     G---5'
EcoRII	Escherichia coli	5'CCWGG 3'GGWCC	5'---     CCWGG---3' 3'---GGWCC     ---5'
BamHI	Bacillus amyloliquefaciens	5'GGATCC 3'CCTAGG	5'---G     GATCC---3' 3'---CCTAG     G---5'
HindIII	Haemophilus influenzae	5'AAGCTT 3'TTCGAA	5'---A     AGCTT---3' 3'---TTCGA     A---5'
TaqI	Thermus aquaticus	5'TCGA 3'AGCT	5'---T   CGA---3' 3'---AGC   T---5'
NotI	Nocardia otitidis	5'GCGGCCGC 3'CGCCGGCG	5'---GC   GGCCGC---3' 3'---CGCCGG   CG---5'
HinfI	Haemophilus influenzae	5'GANTC 3'CTNAG	5'---G   ANTC---3' 3'---CTNA   G---5'
Sau3A	Staphylococcus aureus	5'GATC 3'CTAG	5'---     GATC---3' 3'---CTAG     ---3'
PovII*	Proteus vulgaris	5'CAGCTG 3'GTCGAC	5'---CAG  CTG---3' 3'---GTC  GAC---5'
SmaI*	Serratia marcescens	5'CCCGGG 3'GGGCCC	5'---CCC  GGG---3' 3'---GGG  CCC---5'
HaeIII*	Haemophilus aegyptius	5'GGCC 3'CCGG	5'---GG  CC---3' 3'---CC  GG---5'
AluI*	Arthrobacter luteus	5'AGCT 3'TCGA	5'---AG  CT---3' 3'---TC  GA---5'
EcoRV*	Escherichia coli	5'GATATC 3'CTATAG	5'---GAT  ATC---3' 3'---CTA  TAG---5'
KpnI	Klebsiella pneumoniae	5'GGTACC 3'CCATGG	5'---GGTAC  C---3' 3'---C  CATGG---5'
PstI	Providencia stuartii	5'CTGCAG 3'GACGTC	5'---CTGCA  G---3' 3'---G  ACGTC---5'
SacI	Streptomyces achromogenes	5'GAGCTC 3'CTCGAG	5'---GAGCT  C---3' 3'---C  TCGAG---5'
SalI	Streptomyces albus	5'GTCGAC 3'CAGCTG	5'---G  TCGAC---3' 3'---CAGCT  G---5'
ScaI	Streptomyces caespitosus	5'AGTACT 3'TCATGA	5'---AGT  ACT---3' 3'---TCA  TGA---5'
SphI	Streptomyces phaeochromogenes	5'GCATGC 3'CGTACG	5'---G  CATGC---3' 3'---CGTAC  G---5'
StuI	Streptomyces tubercidicus	5'AGGCCT 3'TCCGGA	5'---AGG  CCT---3' 3'---TCC  GGA---5'
XbaI	Xanthomonas badrii	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

Enzyme Immobilization

ENZYME IMMOBILIZATION

Immobilization means imprisonment of an enzyme in a distinct phase. Immobilized enzymes are enzymes which are attached in or onto the surface of an insoluble support. The immobilized enzymes have several advantages over the soluble enzyme:

Convenience: Miniscule amounts of protein dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.

Economical: Immobilized enzymes can be easily removed from the reaction. There is no loss of enzymes. So the enzymes can be repeatedly reused.

Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

Less contamination: There is less chance of contamination in products while immobilized enzymes are used.

There are a number of requirements to achieve a successful immobilization:

—  The biological component must retain substantial biological activity after attachment

—  It must have a long-term stability

—  The sensitivity of the enzyme must be preserved after attachment

—  Overloading can block or inactivate the active site of the immobilized biomaterial, therefore, must be avoided

Methods of Enzyme Immobilization:

There are many different methods of immobilizing enzymes. The following methods are commonly used;

1. adsorption
2. entrapment
3. encapsulation
4. covalent binding

  

Figure 1: Different methods of enzyme immobilization

1. Adsorption:

It is the simplest immobilization method in which the enzyme and the support are mixed in suitable conditions. The first immobilized enzyme model: invertase on the activated charcoal was developed by Nelson and Griffin, 1916. The forces are weak so leakage is generally a problem in adsorption method. Supports such as alluminium hydroxide are often utilized. With a suitable charged matrix, ionic interactions may also be promoted. This technique is technically undemanding and economically attractive. The regeneration is also easy in this technique. The best known industrial example: amino acylase immobilized on DEAE-Sephadex in the production of amino acids is an example of adsortion method.

2. Entrapment

Enzymes may be entrapped within the matrix of a polymeric gel such as polyacrylamide type gels naturally derived gels e.g. cellulose triacetate, agar, gelatin carrageenan, alginate etc. The form and nature of matrix vary. The pore size of matrix should be adjusted to prevent the loss of enzyme from the matrix due to diffusion.

In order to immobilize the enzymes by this technique, the enzyme together with the gel monomers are incubated. Then gel polymerization is promoted by adding few catalysts. Once there is complete polymerization of gels, the enzymes are immobilized in to the net working of the polymerized gels. Polyacrylamide and polymethacrylamide gels are examples of gels used in  this technique. Gel pore size is a crucial factor in this technique.

Figure 2: Enzyme immobilization by entrapment and encapsulation techniques

3. Encapsulation

Encapsulation involves entrapping the enzymes within a semipermeable membrane capsule such as cellulose nitrate and nylon-based membranes. The method of encapsulation is cheap and simple but its effectiveness largely depends on the stability of enzyme although the catalyst is very effectively retained wiithin the capsule. The main disadvantage of this technique is that only small amount of substrate molecule is utilized with the intact membrane.

4. Covalent binding

The most widely used method for enzyme immobilization is the covalent binding method. It is technically more complex and requires a variety of often expensive chemicals. It is time-consuming. But immobilized enzyme preparations are stable and leaching is minimal. Enzymes are immobilized by a suitable group in the surface such as hydroxyl groups in supports (e.g cellulose, dextran, agarose) and amino, carboxyl and sulfhydryl groups in amino acids. The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding. Therefore, covalent binding may alter the conformational structure and active center of the enzyme, resulting in major loss of activity and/or changes of the substrate. Covalent attachment to a support matrix must involve only functional groups of the enzyme that are not essential for catalytic action. Higher activities result from prevention of inactivation reactions with amino acid residues of the active sites. A number of protective methods have been devised such as covalent attachment of the enzyme in the presence of a competitive inhibitor or substrate . Many factors influence on the activity of enzymes while immmobilizing by this technique. The form, shape, density, porosity, pore size distribution, operational stability and particle size distribution of the supporting matrix will influence the result.

The ideal support should be cheap, inert, physically strong and stable. Ideally, it should:

—  increase the enzyme specificity (kcat/Km)

—  shift the pH optimum to the desired value for the process

—  discourage microbial growth and non-specific adsorption

Some matrices may possess other properties which are useful for particular purposes such as;

—  ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst by means of magnetic fields)

—  a catalytic surface (e.g. manganese dioxide, which catalytically removes the inactivating hydrogen peroxide produced by most oxidases)

—  There is usually a decrease in specific activity of an enzyme upon insolubilization: denaturation caused by the coupling process

—  Microenvironment after immobilization may be drastically different from that existing in free solution: the physical and chemical character of the support matrix, or interactions of the matrix with substrates or products involved in the enzymatic reaction

—  The Michaelis constant may decrease by more than one order of magnitude when substrate of opposite charge to the carrier matrix