Restriction endonucleases: Definition, Types, Nomenclature Unit 2 Notes
An enzyme from bacteria that can recognize specific base sequence / recognition sequence in DNA and cut the DNA at that site (the restriction site).
- Nucleases are of two types:
- Exonucleases – They cut nucleotides at the ends of the DNA.
- Endonucleases – They make cuts at specific positions within the DNA.
- They are also known as molecular scissors and belong to class of enzymes called nucleases.
- Restriction endonuclease was discovered by Hamilton Smith, Werner Arber and Daniel Nathans for which they received Nobel Prize.
- HindII was the first restriction enzyme to be isolated.
- It was obtained from bacterium Haemophilus influenza
- Hundreds of restriction enzymes have been identified produced by different microbial species.
- Each of them has their own restriction site.
- These enzymes cut the recognized sequence by hydrolysis.
- Most recognize and cut palindromic sequences. A word which spells the same when read from backward or forward is called a pallindrome, e.g. MALAYALAM, palindrome in a DNA is a sequence of base pairs that reads the same on two complementary strands, eg. GAATTC- complementary base pairs will be CTTAAG.
- After cutting the DNA sequence with restriction enzymes, sticky/staggered ends are created which are essential for proper attachment of desired gene.
- By choosing correct restriction enzyme we can cut DNA exactly at the position where the gene of interest has to be inserted.
- RE’s destroy non-self DNA (DNA of other organisms e.g. viral DNA is destroyed by bacteria, by cutting it due to methylase activity).
- They are naturally present in micro-organism which is used for their defense mechanism i.e. to restrict the entry of viruses; hence the name restriction is given.
- They have the ability to cut foreign genes.
- Methylase enzyme
- In bacteria there are enzymes called as methylase enzymes.
- These enzymes recognize self DNA sequence i.e. bacteria and add methyl group on it and hence methylates the sequence.
- This methylated sequence is protected and not destroyed by bacterial RE’s and hence can be differentiated from foreign DNA and protected.
- Bacteria does not methylate the foreign DNA (non self DNA) and hence foreign DNA is destroyed by RE’s produced by bacteria
Note: Enzymes are termed as restriction because it restricts the entry of foreign DNA, multiplication of foreign DNA and also it prevents the replication of phage DNA
Types of Restriction endonucleases
They are three main types of restriction endonuclease
- Type I
- Type II
- Type III
They differ from each other in their mode of action.
Type II restriction enzymes are used in r-DNA technology because they can be used in vitro to identify and cleave within specific DNA sequences usually having 4-8 nucleotides.
More than 350 different type II endonucleases with 100 different recognition sequences are known.
Type I and type III (Exonucleases)
- They are similar in their action.
- The restriction activity and methylase activity is present in a single large enzyme complex.
- They will cut at few base pairs away from the restriction site even after identifying the same sequence. Hence called as exonucleases.
Type II (Endonucleases)
- The restriction activity and methylase activity are different i.e. they are not present in single enzyme complex.
- Type II RE’s will identify the sequence in a DNA and cut within sequence at that specific site called as restriction site. Hence the name endonucleases.
- Widely used in recombinant DNA technology, as here the need is to cut DNA at a particular site.
Restriction endonucleases are named by a standard procedure, with particular reference to the bacteria from which they are isolated. The first letter (in italics) of the enzymes indicates the genus followed by the first two letters (also in italics) of the species, then comes the strain of organism and finally a roman numerical indicating the order of discovery. A couple of examples are given below
- EcoR I is from Escherichia (E) coli (co) strain Ry 13 (R) and first endonuclease (I) to be discovered.
- Hind III is from Haemophilus (H) influenzae (in) strain Rd (d) and the third endonuclease (III)
- BamH1 is from Bacillus (B) amyloliquefaciens (am) strain (H) and is the 1st enzyme
Recognition site is the site where the DNA is cut by restriction endonucleases. Each restriction enzyme is highly specific, recognizing a particular short DNA sequence, or restriction site, and cutting both DNA strands at the same time at specific points within that site. Most restriction sites are palindromic.
Restriction Enzymes Indicating the Restriction Site
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