Restriction enzyme, Restriction endonuclease, Molecular scissors is a protein produced by bacteria that recognize and cleave a short, specific sequence of nucleotide bases of the linear double-stranded DNA molecule. Restriction enzymes are found in many strains of bacteria and their main function is to prevent and restrict any foreign DNA by destroying it and can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA. Over 3000 restriction endonucleases have been studied in detail, and more than 600 of these are available commercially.
Recognition site or restriction sites
Restriction endonucleases recognize a specific sequence of nucleotides (usually4-8 base pairs in length) and produce a double-stranded cut in the DNA. These are generally palindromic sequences and the number of bases in the sequence will determine how often the site will appear by chance in any given genome, e.g., a 4 bases sequence would occur once every 4^4 or 256bp, 6 bases, 4^6 or 4,096bp, and 8 bases would be 4^8 or 65,536bp.
Restriction enzymes recognize a palindromic recognition site reads the same on the reverse strand as it does on the forward strand when both are read in the same orientation. In theory, there are two types of palindromic sequences that can be possible in DNA.
- Mirror-like palindrome – a sequence reads the same forward and backward on a single strand of DNA, as in GTAATG.
- Inverted repeat palindrome is also a sequence that reads the same forward and backward, but the forward and backward sequences are found in complementary DNA strands. Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.
Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation. For example, the restriction enzyme EcoRI recognizes the palindromic sequence GAATTC and cuts between the G and the A on both the top and bottom strands, leaving an overhang known as a sticky end on each end of AATT.
Some restriction enzymes, for example, SmaI cut DNA at a restriction site in a manner which leaves no overhang, called a blunt end.
Different restriction enzymes that recognize the same sequence but often in different locations are known as neoschizomers. The enzymes that recognize and cleave in the same location are known as isoschizomers.
Naturally occurring restriction endonucleases are classified into four groups based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. These are designated as Type I, Type II, and Type III and Type IV enzymes.
• Type I enzymes (EC 126.96.36.199) – are complex, multisubunit, combination restriction-and-modification enzymes that cleave DNA at sites remote from (at least 1000 bp) their recognition site Type I restriction endonuclease require ATP and S-adenosyl-L-methionine and magnesium (Mg2+) ions for their activity. Type I enzymes are of considerable biochemical interest, but they have little practical value since they do not produce discrete restriction fragments or distinct gel-banding patterns.
• Type II enzymes (EC 188.8.131.52)- They recognize and cleave DNA within or at short specific distances from a recognition site. They do not use ATP or AdoMet for their activity—they usually require only Mg2+ as a cofactor. They are single function (restriction) enzymes independent of methylase.
• Type III enzymes (EC 184.108.40.206) –are also a combination restriction-and-modification enzymes. They recognize two separate non-palindromic sequences that are inversely oriented.They cleave at about 20–30 base pairs from a recognition sequence and rarely give complete digests. They need ATP (but do not hydrolyze it). S-adenosyl-L-methionine stimulates the reaction but is not required.
• Type IV enzymes – they recognize modified DNA, e.g. methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA
Artificial Restriction Enzymes
In addition to natural restriction enzymes, it is now possible to synthesize artificial restriction enzymes that cut DNA at any desired sequence. Examples:
• zinc-finger nucleases
• CRISPR RNA molecules
- clone a particular fragment into a plasmid/ phagemid/ artificial chromosome
- The different fragments of DNA generated by restriction enzymes also produce a specific pattern of bands after gel electrophoresis and can be used for DNA fingerprinting.
- Can be used to Understand Epigenetic Modifications, polymorphisms
- Compare DNA from two or more samples for testing similarity i.e. restriction fragment length polymorphism (RFLP).
- Restriction enzymes can be used generate DNA map that can give the relative positions of the genes.
- Restriction enzymes can be used to Construct DNA Libraries
- Restriction enzymes can be used to create Nicks in DNA