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🧬 Restriction enzymes

Restriction enzymes.

This lesson explains the core ideas, methods, and exam-relevant applications for this topic in plant biotechnology. Focus on definitions, process steps, and practical uses for revision.

Enzymes used in Genetic Engineering

The ability to manipulate DNA in vitro depends entirely on the availability of purified

enzymes that can cleave, modify and join the DNA molecule in specific ways.

At present, no chemical method can achieve the ability to manipulate the DNA in vitro in

a predictable way. Only enzymes are able to carry out the function of manipulating the

DNA. Each enzyme has a vital role to play in the process of genetic engineering. The

various enzymes used in genetic engineering are as follows:

 Nucleases

 Restriction enzymes

 DNA ligase

 Kinase

 Phosphatase

 Reverse transcriptase

 Terminal Deoxynucleotide Transferase

 RNaseP

The functions of the various enzymes used in genetic engineering are depicted below:

Nucleases - Nucleases are a group of enzymes which cleave or cut the genetic material

(DNA or RNA).

DNase and RNase

Nucleases are further classified into two types based upon the substrate on which they

act. Nucleases which act on or cut the DNA are classified as DNases, whereas those

which act on the RNA are called as RNases.

DNases are further classified into two types based upon the position where they act.

DNases that act on the ends or terminal regions of DNA are called as exonucleases and

those that act at a non-specific region in the centre of the DNA are called as

endonucleases.

Exonucleases require a DNA strand with at least two 5 and 3 ends. They cannot act on

DNA which is circular. Endonucleases can act on circular DNA and do not require any

free DNA ends ( i.e., 5 or 3 end). Exonucleases release nucleotides (Nucleic acid + sugar

  • phosphate), whereas endonucleases release short segments of DNA.

DNases which act on specific positions or sequences on the DNA are called as

restriction endonucleases. The sequences which are recognized by the restriction

endonucleases or restriction enzymes (RE) are called as recognition sequences or

restriction sites. These sequences are palindromic sequences. Different restriction

enzymes present in different bacteria can recognize different or same restriction sites.

But they will cut at two different points within the restriction site. Such restriction

enzymes are called as isoschizomers. Interestingly no two restriction enzymes from a

single bacterium will cut at the same restriction site.

Mode of action

The restriction enzyme binds to the recognition site and checks for the methylation

(presence of methyl group on the DNA at a specific nucleotide). If there is methylation in

the recognition sequence, then, it just falls off the DNA and does not cut. If only one

strand in the DNA molecule is methylated in the recognition sequence and the other

strand is not methylated, then RE (only type I and type III) will methylate the other strand

at the required position. The methyl group is taken by the RE from S-adenosyl

methionine by using modification site present in the restriction enzymes.

However, type II restriction enzymes take the help of another enzyme called methylase,

and methylate the DNA. Then RE clears the DNA. If there is no methylation on both the

strands of DNA, then RE cleaves the DNA.

It is only by this methylation mechanism that, RE, although present in bacteria, does not

cleave the bacterial DNA but cleaves the foreign DNA. But there are some restriction

enzymes which function exactly in reverse mode. They cut the DNA if it is a methylate.

Star activity

Sometimes restriction enzymes recognize and cleave the DNA strand at the recognition

site with asymmetrical palindromic sequence, for example Bam HI cuts at the sequence

GA TCC, but under extreme conditions such as low ionic strength it will cleave in any of

the following sequence NGA TCC, GPOA TCC, GGNTCC. Such an activity of the RE is

called star activity.

Nomenclature of Restriction Enzymes

As a large number of restriction enzymes have been discovered, a uniform

nomenclature system is adopted to avoid confusion. This nomenclature was first

proposed by Smith and Nattens in 1973.

Every restriction enzyme would have a specific name which would identify it uniquely.

The first three letters, in italics, indicate the biological source of the enzymes, the first

letter being the initial of the genus and the second and third being the first two letters of

the species name. Thus restriction enzymes from Escherichia coli are called Eco;

Haemophilus influenzae becomes Hin; Diplocococcus pneumoniae Dpn and so on. Then

comes a letter that identifies the strain of bacteria; Eco R for strain R. Finally there is a

roman numeral for the particular enzyme if there are more than one in the strain in

question; Eco RI for the first enzyme from E. coli R, Eco RII for the second.

Types

The restriction endonucleases can be divided into three groups as type I, II and III.

Types I and III have an ATP dependent restriction activity and a modification activity

resident in the same multimeric protein. Both these types recognize unmethylated

recognition sequences in DNA. Type I enzymes cleave the DNA at random site, whereas

Type III cleave at a specific site. Type II restriction modification system possess separate

enzymes for endonuclease and methylase activity and are the most widely used for

genetic manipulation.

Type I Restriction Enzymes

These restriction enzymes recognize the recognition site, but cleave the DNA

somewhere between 400 base pairs (bp) to 10,000 bp or 10 kbp right or left. The

cleavage site is not specific. These enzymes are made up of three peptides with multiple

functions. These enzymes require Mg++, ATP and S adenosyl methionine for cleavage

or for enzymatic hydrolysis of DNA. These enzymes are studied for general interest

rather than as useful tools for genetic engineering.

Type II Restriction Enzymes

Restriction enzymes of this type recognize the restriction site and cleave the DNA within

the recognition site or sequence. These enzymes require Mg++ as cofactor for cleavage

activity and can generate 5 -PO4 or 3 -OH. Enzymes of this type are highly important

because of their specificity. Type II restriction enzymes are further divided into two types

based upon their mode of cutting .

Type II Restriction Enzymes - Blend End Cutters

Blunt end cutters Type II restriction enzymes of this class cut the DNA strands at same

points on both the strands of DNA within the recognition sequence. The DNA strands

generated are completely base paired. Such fragments are called as blunt ended or

flush ended fragments.

Type II Restriction Enzymes - Cohesive End Cutters

Cohesive end cutter Type II restriction enzymes of this class cut the DNA stands at

different points on both the strands of DNA within the recognition sequence. They

generate a short single-stranded sequence at the end. This short single strand sequence

is called as sticky or cohesive end. This cohesive end may contain 5 -PO 4 or 3 -OH,

based upon the terminal molecule (5 -PO4 or 3 -OH). These enzymes are further

classified as 5end cutter (if 5 -PO 4 is present) or 3 -end cutter (if3' -OH is present) .

Type III Restriction Enzymes

Type III Restriction enzymes of this type recognize the recognition site, but cut the DNA

1 kbp away from the restriction site. These enzymes are made up of two peptides or

subunits. These enzymes require A TP, Mg++ and S-adenosyl methionine for action.

The properties of three types of restriction endonucleases and a list of enzymes are

given below.

Property Type I Type II Type III
Structure Enzyme complex of 500-
600 k dal composed of
three separate subunits
Normally
homodimers
of 20-70 k dal		 Heterodimers
with
subunits of 70 and 100 k
dal
Composition Multienzyme
complex
with R (endonuclease),
M (methylase) and S
(specificity) subunits
Separate
enzymes;
endonuclease
is
a
homodimer, methylase
a monomer		 M
subunit
provides
specificity on its own;
functions as methylase;
as heterodimer with R
subunit;
functions
as
methylase-
endonuclease
Cofactors Mg2+, ATP, S-
adenosylmethionine
(SAM) (needed for
cleavage as well as
methylation)		 Mg2+, SAM (for
methylation only)		 Mg2+, ATP (for
cleavage), SAM (needed
for methylation: stimulate
cleavage)
Recognition sites Asymmetric,
bipartite,
may be degenerate; 13-
15 base pairs containing
interruption of 6 to 8 base
pairs
Asymmetric,
may
be
bipartite,
may
be
degenerate; 4 to 8 base
pairs
normally
180°
rotational symmetry

Asymmetric,
uninterrupted,
5-6
nucleotide long with no
rotational symmetry
Cleavage Non-specific,
variable
distance
(100-1000
nucleotides)
from
recognition site		 Precise cleavage within
recognition
site
at
defined distance		 Precise cleavage at a
fixed
distance;
25-27
nucleotides
from
recognition site
Example _Eco_K _Eco_RI _Eco_P1

Some restriction endonucleases and their recognition sites

Enzyme Recognition site Enzyme Recognition site
4-base cutters 4-base cutters 6-base cutters 6-base cutters
Mbo_I, Dpn_I,_ Sau_3AI
/GATC
Msp_I, Hpa_II
C/CGG
_Alu_I
AG/CT
_Hae_III
GG/CC
_Tai_I
AC/GT		 Mbo_I, Dpn_I,_ Sau_3AI
/GATC
Msp_I, Hpa_II
C/CGG
_Alu_I
AG/CT
_Hae_III
GG/CC
_Tai_I
AC/GT		 _Bgl_I
A/GATCT
_Cla_I
AT/CGAT
_Pvu_II
CAG/CTG
_Pvu_I
CGAT/CG		 _Bgl_I
A/GATCT
_Cla_I
AT/CGAT
_Pvu_II
CAG/CTG
_Pvu_I
CGAT/CG
Mbo_I, Dpn_I,_ Sau_3AI
/GATC
Msp_I, Hpa_II
C/CGG
_Alu_I
AG/CT
_Hae_III
GG/CC
_Tai_I
AC/GT		 Mbo_I, Dpn_I,_ Sau_3AI
/GATC
Msp_I, Hpa_II
C/CGG
_Alu_I
AG/CT
_Hae_III
GG/CC
_Tai_I
AC/GT

8-base cutters

8-base cutters
Mbo_I, Dpn_I,_ Sau_3AI
/GATC
Msp_I, Hpa_II
C/CGG
_Alu_I
AG/CT
_Hae_III
GG/CC
_Tai_I
AC/GT		 Mbo_I, Dpn_I,_ Sau_3AI
/GATC
Msp_I, Hpa_II
C/CGG
_Alu_I
AG/CT
_Hae_III
GG/CC
_Tai_I
AC/GT		 _Not_I
GC/GGCCCC
_Sbf_I
CCTGCA/GG		 _Not_I
GC/GGCCCC
_Sbf_I
CCTGCA/GG

DNA Ligase

Recombinant DNA experiments require the joining of two different DNA segments or

fragments in vitro . The cohesive ends generated by some RE will anneal themselves by

forming hydrogen bonds. But the segments annealed thus are weak and do not

withstand experimental conditions. To get a stable joining, the DNA should be joined by

using an enzyme called ligase. DNA ligase joins the DNA molecule covalently by

catalysing the formation of phosphodiester bonds between adjacent nucleotides.

DNA ligase isolated from E. coli and T 4 bacteriophage is widely used. These ligases

more or less catalyse the reaction in the same way and differ only in requirements of

cofactor. T4 ligase requires ATP as cofactor and E. coli ligase requires NADP as

cofactor. The cofactor is first split (ATP - AMP + 2Pi) and then AMP binds to the enzyme

to form the enzyme-AMP complex. This complex then binds to the nick or breaks (with 5'

-PO4 and 3' -OH) and makes a covalent bond in the phosphodiester chain. The ligase

reaction is carried out at 4 [0] C for better results.

Kinase

Kinase is the group of enzymes, which adds a free pyrophosphate (PO4) to a wide

variety of substrates like proteins, DNA and RNA. It uses ATP as cofactor and adds a

phosphate by breaking the ATP into ADP and pyrophosphate. It is widely used in

molecular biology and genetic engineering to add radiolabelled phosphates.

Alkaline phosphatases

Phosphatases are a group of enzymes which remove a phosphate from a variety of

substrates like DNA, RNA and proteins. Phosphatases which act in basic buffers with pH

8 or 9 are called as alkaline phosphatases. Most commonly bacterial alkaline

phosphatases (BAP), calf intestine alkaline phosphatases (CIAP) and shrimp alkaline

phosphatases are used in molecular cloning experiments. The PO4 from the substrate is

removed by forming phosphorylated serine intermediate. Alkaline phosphatase is

metalloenzymes and has Zn++ ions in them.

BAP (bacterial alkaline phosphatase) is a dimer containing six Zn++ ions, two of which

are essential for enzymatic activity. BAP is very stable and is not inactivated by heat and

detergent.

Calf intestine alkaline phosphatase (CIAP) is also a dimer. It requires Zn++ and Mg++

ions for action. CIAP is inactivated by heating at 70 [0] C for twenty minutes or in the

presence of 10 mM EGTA. Alkaline phosphatases are used to remove the PO4 from the

DNA or as reporter enzymes.

Reverse Transcriptase

This enzyme uses an RNA molecule as template and synthesizes a DNA strand

complementary to the RNA molecule. These enzymes are used to synthesize the DNA

from RNA. These enzymes are present in most of the RNA tumour viruses and

retroviruses. Reverse transcriptase enzyme is also called as RNA dependent DNA

polymerase. Reverse transcriptase enzyme, after synthesizing the complementary

strand at the 3 end of the DNA strand, adds a small extra nucleotide stretch without

complementary sequence. This short stretch is called as R-loop.

Terminal Deoxynucleotide transferase

Terminal deoxynucleotide transferase is a polymerase which adds nucleotides at 3' -OH

end (like Klenow fragment) but does not require any complementary sequence and does

not copy any DNA sequence (unlike Klenow fragment). Terminal deoxynucleotide

transferase (TDNT) adds nucleotide whatever comes into its active site and it does not

show any preference for any nucleotide.

RNase P

It specifically cleaves at the 5’ end of RNA. It is a complex enzyme consisting of small

protein (20 kilodaltons) and a 377 -nucleotide RNA molecule. It has been observed that

the RNA molecule possesses at least part of the enzymatic activity of the complex.

Hence, it is an example of ribozyme.

Klenow fragment

E. coli DNA polymerase I consists of a single polypeptide chain. Pol I carry out three

enzymatic reactions that are performed by three distinct functional domains. Two

fragments are obtained when DNA pol I is treated with trypsin/subtilisin in mild

conditions. The larger fragment is called as Klenow fragment. This fragment is 602

amino acids in length. The function of the Klenow fragment is to add nucleotides to the 3

end and 3 -5 exonuclease activity.

Klenow fragment adds nucleotides by using complementary strand as reference. It

cannot extend the DNA without the presence of the complementary strand. If any

nucleotide is added by mistake and the base pair is wrong (i.e., if A is paired to G

instead of T) then by using 3 -5 exonuclease activity present in Klenow fragment, this

mispaired base pair is removed. In general the Klenow fragment has 5 -3 polymerase

and exonuclease activity.

Questions

  1. The enzymes used in rDNA technology includes with star activity is/are ……..

a). Nucleases b). Restriction enzymes c). DNA ligase d).All the above

  1. The enzymes used in rDNA technology includes with star activity is/are ……..

a). Kinase b). Phosphatase c). Reverse transcriptase d).All the above

  1. The enzymes used in rDNA technology includes with star activity is/are ……..

a). Terminal Deoxynucleotide Transferase

b). RNaseP

c). Reverse transcriptase d).All the above

  1. The group of enzymes used in rDNA technology which cleave or cut the genetic

material ……..

a). Nucleases b). RNaseP c). Reverse transcriptase d). All the above

  1. Nucleases act on ……..

a). DNA b). RNA c). Both a and b d). None of the above

  1. Nucleases act on ……..

a). DNA b). RNA c). Both a and b d). None of the above

  1. The nomenclature of restriction enzymes was proposed by ……..

a). Smith b). Nattens c). Both a and b d). None of the above

  1. The restriction enzymes are grouped into ………… types

a). 3 b). 5 c). 2 d).None of the above

  1. The restriction enzymes used widely in rDNA technology is ……..

a). Type I b). Type II c). Type III d).None of the above

  1. The restriction enzyme with star activity is/are ……..

a). Eco Rl b). Bam HI

c). Sal 1 d).All the above

  1. DNA ligase joins the DNA molecule covalently by catalysing the formation of

…………… bonds between adjacent nucleotides.

a). Phosphodiester b). Phosphotriester c). Both a and b d). None of the above

  1. DNA ligase isolated is from ……….

a). E. coli b). T 4 bacteriophage c). Both a and b d). None of the above

  1. DNA ligase isolated from and is widely used.

a). E. coli b). T 4 bacteriophage c). Both a and b d). None of the above

  1. Reverse transcriptase isolated from and is widely used.

a). E. coli b). T 4 bacteriophage c). Both a and b d). None of the above

  1. Rnase P specifically cleaves at the ……… of RNA.

a). 5’ end b). 3’ end c). Both a and b d). None of the above

Summary Cheat Sheet

Quick Recall Points

  • Define key terms in one line and revise their use in plant biotechnology.
  • Memorize major steps, methods, and applications covered in this lesson.
  • Practice exam-style distinctions between related concepts and techniques.

Exam Traps

  • Do not confuse similar terms without checking context and biological level.
  • Revise process order carefully; sequence-based questions are common.
  • Link each method with its most likely application question.

References

1 source • [1]

[1]

Standard BSc Agriculture Plant Biotechnology notes

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