Restriction Enzymes

Course Name : B.Sc Zoology(H)

Semester : V

Paper-DSE2: Animal Biotechnology

Teacher: Samita Maity

Learning Objective

After studying this unit, you shall be able to:

• Understand what a restriction endonuclease (restriction enzyme) is and how it functions.
• Identify restriction enzyme(s) recognition sites given a section of DNA.
• Understand the difference between enzymes that cut double-stranded DNA to produce a region of single-stranded DNA and those that do not, and the appropriate use of each type of enzyme.
• Determine which restriction enzyme to use to create a desired piece of recombinant DNA

​

2

What Is Restriction Enzyme?

• Restriction Enzyme , also called restriction endonuclease,a protein produced by bacteria that recognize specific base sequences in DNA and cut the DNA at that site.
• In the bacterial cell, restriction enzymes cleave foreign DNA ,thus eliminating infecting organism.
• The restriction enzymes are also called molecular scissors.
• Restriction Enzyme , also called restriction endonuclease,a protein produced by bacteria that recognize specific base sequences in DNA and cut the DNA at that site.
• In the bacterial cell, restriction enzymes cleave foreign DNA ,thus eliminating infecting organism.
• The restriction enzymes are also called molecular scissors.

​

​

3

Biological Function Of Restriction Enzyme

• Most bacteria uses restriction Enzyme as a defense against bacteriophages.
• Restriction Enzyme prevent the replication of phage by cleaving its DNA at specific site.
• The host DNA is protected by methylase which add methyl groups to adenine and cytosine bases with in the recognition site thereby modifying the site and protecting the DNA.

​

4

Nomenclature Of Restriction Enzyme

The restriction enzymes are named based on some of the following principle:

• Name of the organism is identified by the first letter of genus name and the first two letter of the species name to form a three letter abbreviation in the italic.

Example: E.coli =Eco.

• A strain or type identified as subscript.

Example: EcoK for E.coli strain k.

• In such case where the restriction and modification system are genetically specified by a virus or plasmid, the extra chromosomal element is identified by a subscript.

Example: EcoRI, EcoPI.

• When an organism produce more than one enzyme ,they are identified by Roman numerals.

Example: HindI, HindII , Hind III.

​

​

5

Types Of Restriction Enzyme

There are three distinct type of Restriction Enzyme:

• Type I Restriction Enzyme
• Type II Restriction Enzyme
• Type III Restriction Enzyme

​

These type are categorized based on:

• Their composition
• Enzyme cofactor requirement.
• The nature of the target sequence
• Position of their DNA cleavage site

​

6

Type I Restriction Enzyme

• They are complex nucleases which function simultaneously as an Endonuclease and a methylase .
• They move along the DNA in a reaction and require Mg⁺⁺, S-adenosly methionine and ATP as co factor.
• They are single multifunctional enzymes consisting of three different subunits:
• Restriction sub-unit
• Modification sub-unit
• Specificity sub-unit
• They recognize specific sites within the DNA but do not cleave at these sites.
• Restriction site of Type I enzyme in 15 bp long and cleavage site is about 1000 bp away from the 5’- end of the recognition sequence TCA which is present within 15 bp long recognition site.

Example: EcoK.

​

​

7

Type II Restriction Enzyme

• They are simple endonucleases consisting of single polypeptide chain and required no ATP for DNA degradation.
• They have separate activities of cleaving and methylation.
• They are most stable enzyme require Mg⁺⁺ as a co-factor.
• Type II restriction enzyme make cuts only with in the restriction site and produce two single stranded break.
• Recognition sites are the palindromes or palindromic sequence where most Type II restriction enzyme bind and cut the DNA molecule.
• (Palindromes are the nucleotide pair sequences that are same when read forward or backward from a central axis of the symmetry i.e. two stand are identical when both are read from the same polarity i.e. in 5’-3’direction.)
• The length of recognition sites of different enzyme varies which can be four ,five ,six eight or more nucleotide pairs.s
• The properties of Type II restriction enzymes vary with its specificity i.e. its changes with the reaction conditions such as lowering the NaCl concentration ,increasing temperature, replacing Mg⁺⁺ with Mn⁺⁺.
• Daniel Nathans first used these enzymes for mapping and analysis of gene and genomes.

Example- Eco RI

​

8

Examples of Type II Restriction Enzyme

9

Type III Restriction Enzyme

• The Type III enzyme is made up of two sub units, one specifies for site recognition and modification the other for cleavage.
• In a reaction, they move along the DNA and require ATP as source of ATP and Mg++ as co-factor.
• They have symmetrical recognition sites and cleave DNA at specific non-palindromic sequence.
• They cleave the DNA in the immediate vicinity of their recognition site.

Example: EcopI,Ecop15.

10

Cleavage Pattern Of Type II Endonuclease

• Most of the Type II restriction Endonuclease cleaves the DNA molecule within their specific recognition sequences, but some produces cuts immediately outside the target sequence.
• Example: NlaIII Sau3A.

​

These cuts are two types-

• Staggered Cuts
• Blunt ended Cuts

​

​

11

Staggered Cut Restriction Enzyme

Staggered Cut

• Most of the enzyme produces staggered cuts in which two strand of a DNA double helix are cleaved at different location.
• This generate protruding ends (3’ or 5’) i.e. one strand of the double helix extends some bases beyond the other.
• Due to the palindromic nature of the target sites ,the two protruding end generated by such a cleavage by a given enzyme have complementary base sequence.
• As a result ,they readily pair with each other such end are called cohesive or sticky end.
• An important consequence of this fact is that when fragments generated from single restriction enzyme from different DNAs are mixed, they joined together due to their sticky end.

12

Staggered Cut Restriction Enzyme

Staggered Cut

• Most of the enzyme produces staggered cuts in which two strand of a DNA double helix are cleaved at different location.
• This generate protruding ends (3’ or 5’) i.e. one strand of the double helix extends some bases beyond the other.
• Due to the palindromic nature of the target sites ,the two protruding end generated by such a cleavage by a given enzyme have complementary base sequence.
• As a result ,they readily pair with each other such end are called cohesive or sticky end.
• An important consequence of this fact is that when fragments generated from single restriction enzyme from different DNAs are mixed, they joined together due to their sticky end.

13

Blunt End Restriction Enzyme

Some restriction Enzymes, cut both the strands of DNA molecule at the same site so that the resulting termini or ends have blunt ends in which the two strands end at the same point.

​

14

Isoschizomers and Neoschizomers

Isoschizomers:

• Restriction Enzymes that have the same recognition sequence as well as the same cleavage site are caller Isoschizomers.

​

Neoschizomers:

• Restriction enzyme that have the same recognition sequence but cleave the DNA at different site within that sequence are called Neoschizomers.

​

​

15

Uses Of Restriction Enzymes

• Recombinant DNA Technology
• DNA Sequencing
• cDNA/ Genomic Library Construction
• DNA Mapping
• RFLP Analysis (Restriction Fragment Length Polymorphism)

16

Comparison Between Different Types Of Restriction Enzyme

17

Thank You

18