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RESTRICTION ENDONUCLEASE ,TYPES , MECHANISM , APPLICATION

Restriction endonucleases are enzymes that cut DNA at specific internal sites within the molecule. They are naturally produced by bacteria and archaea as part of a protective system against invading foreign DNA, such as bacteriophages and plasmids. When foreign DNA enters a bacterial cell, restriction endonucleases recognize particular nucleotide sequences and cleave the DNA, thereby preventing the establishment of infection. To protect their own genomic DNA from cleavage, bacteria modify it by methylation through companion enzymes called DNA methyltransferases. Together, restriction endonucleases and methyltransferases form the restriction–modification system. Restriction endonucleases recognize short, specific DNA sequences known as recognition sites, which are usually 4 to 8 base pairs long. These recognition sequences are often palindromic in nature, meaning the sequence reads the same in the 5′ to 3′ direction on both strands of the DNA. Once the enzyme binds to its recognition site, it cuts the phosphodiester backbone of the DNA at or near that site. Depending on the enzyme, the cleavage may result in sticky ends, which have short single-stranded overhangs, or blunt ends, which have no overhangs. Sticky ends are particularly useful in molecular cloning because they can easily anneal with complementary sequences. Based on their structural features, recognition patterns, cleavage position, and cofactor requirements, restriction endonucleases are broadly classified into several types, mainly Type I, Type II, and Type III. Type I restriction enzymes recognize specific DNA sequences but cut the DNA at sites far away from the recognition sequence. They require ATP, S-adenosyl methionine, and magnesium ions for their activity. Type III restriction enzymes cleave DNA at a short distance from the recognition site and require ATP and magnesium ions. Type II restriction endonucleases are the most extensively studied and widely used enzymes in molecular biology. They cleave DNA within or very close to their recognition sequences and generally require only magnesium ions as cofactors, making them highly predictable and reliable tools. Type II restriction enzymes are named based on the bacterial species from which they are isolated. For example, EcoRI is derived from Escherichia coli, while HindIII comes from Haemophilus influenzae. Each enzyme has a unique recognition sequence and cleavage pattern. This specificity allows scientists to cut DNA molecules at defined positions, producing reproducible fragments of known sizes. Restriction endonucleases play a central role in recombinant DNA technology. They are essential for gene cloning, where both the gene of interest and the vector DNA are cut with the same restriction enzyme to generate compatible ends. They are also used in DNA mapping, genome analysis, restriction fragment length polymorphism studies, and diagnostic applications. In addition, restriction enzymes are valuable tools in genetic engin

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RESTRICTION ENDONUCLEASE REMYA M S ASSISTANT PROFESSOR DEPARTMENT OF MICROBIOLOGY ST.MARY’S COLLEGE ( AUTONOMOUS ) , THRISSUR
INTRODUCTION  Restriction endonucleases or restriction enzymes are enzymes that recognize a specific sequence of nucleotides and cleave DNA at specific nucleotide sequences.  Restriction endonucleases cut the DNA double helix in very precise ways. It cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites.  The sequence recognized is often 4, 6 or 8 nucleotide long.  Eg. Eco RI recognizes the sequence GAATTC and cut between G and A. Restriction endonucleases are also called molecular scisssors.
SOURCE OF RESTRICTION ENZYMES  The natural source of restriction endonucleases are bacterial cells.  These enzymes are called restriction enzymes because they restrict infection of bacteria by certain viruses (i.e., bacteriophages), by degrading the viral DNA without affecting the bacterial DNA. Thus, their function in the bacterial cell is to destroy foreign DNA that might enter the cell.  The restriction enzyme recognizes the foreign DNA and cuts it at several sites along the molecule.  Each bacterium has its own unique restriction enzymes and each enzyme recognizes only one type of sequence.
NOMENCLATURE  The species name of the host organism is identified by the first letter of the genus name and the first two letters of the specific epithet to generate a three letter abbreviation. This abbreviation is always written in italics.  Where a particular strain has been the source then this is identified.  When a particular host strain has several different R-M systems, these are identified by roman numerals. E – Escherichia: Genus co- coli: specific species R- RY13: strain I- First identified: order of identification in the bacterium
RECOGNITION SITES  The DNA sequences recognized by restriction enzymes .  Most of the recognition sequences are palindromic.  Palindromes are the base sequences that read the same on the two strands but in opposite directions.  Two types of palindromic sequences 1. Mirror like palindrome It is similar to those found in ordinary text , in which a sequence read the same forward and backward on a single strand of DNA Eg : GTAATG 2. The inverted repeat palindrome Palindromes are the base sequences that read the same on the two strands but in opposite directions.
 For example, if the sequence on one strand is GAATTC read in 5’→3′ direction, the sequence on the opposite strand is CTTAAG read in the 3’→5′ direction, but when both strands are read in the 5’→ 3′ direction the sequence is the same. 5′ GAATTC 3′ 3′ CTTAAG 5′
MECHANISM OF CLEAVAGE OF RESTRICTION ENZYMES  When a restriction endonuclease recognizes a particular sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides.  To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
NATURE OF CUTS  enzymes cut straight across the molecule at the symmetrical axis producing blunt ends.  restriction enzymes that cut between the same two bases away from the point of symmetry on two strands, thus, producing a staggering break called sticky end
TYPES OF RESTRICTION ENDONUCLEASE  RE mainly classified into four different types based on the subunit composition , cleavage position, sequence specificity and co factor requirement  Type I: This system is also known as restriction and modification system.  They function both as endonuclease and methylase and these are enzymes with three different subunits each for recognition, cleavage and methylation.  These enzymes require Mg ++ , S-adenosyl methionine and ATP as cofactors. Recognizes a single sequence of 15 bp length and cleaves at a site up to 1000 bp away.  The type I systems were the first to be characterized. The active enzyme consists of two restriction subunits, two modification (methylation) subunits and one recognition subunit.  The enzyme cuts unmodified DNA at a distance from the recognition sequence Eg. Eco B and Eco K
 Type II: these are simple consisting of single polypeptide chain and do not require ATP for cleaving DNA.  They are highly stable and require Mg ++ .  The first isolated type II restriction endonuclease was from E.coli strain RY and it was termed Eco RI.  Type II enzymes recognize the target sequence and cleave or modify the DNA at the same site.  Restriction and modification are mediated by separate enzymes and so it is possible to cleave DNA in the absence of modification.  The restriction activities or cleavage do not require cofactors such as ATP or S-adenosylmethionine, making them easier to use.  Many of them make a staggered break in the DNA.  The properties of Type II restriction enzymes can be varied with changes in reaction conditions such as NaCl concentration, temperature, presence of Mg ++ or Mn ++ , etc.  Eg : Hae III , Bam HI , Sau 3AI
 Type III: these are enzymes with two different subunits, one for recognition and modification and one for cleavage.  Require ATP and Mg ++. Recognizes and methylates same sequence but cleaves 24–26 bp away.  Type III enzymes have symmetrical recognition sequences but otherwise resemble type I systems and are of little value in genetic engineering.  They recognize two separate non-palindromic sequences and cut the DNA 20–30 bp away from the recognition site.  Eg. Mbo II, Fok I, Hpa I  Type IV : These enzymes specifically target modified DNA (methylated, hydroxymethylated, or glucosyl- hydroxymethylated DNA).  They do not cut "normal" unmethylated DNA.
APPLICATIONS OF RESTRICTION ENZYMES  Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason, they are indispensable tools of recombinant DNA technology (genetic engineering).  The most useful aspect of restriction enzymes is that each enzyme recognizes the same unique base sequence regardless of the source of the DNA. It means that these enzymes establish fixed landmarks along an otherwise very regular DNA molecule. This allows dividing a long DNA molecule into fragments that can be separated from each other by size with the technique of gel electrophoresis.  Each fragment, thus generated, are also available for further analysis, including the sequencing.  One value of cutting DNA molecule up into discrete fragments is being able to locate a particular gene on the fragment where it resides which is done by the general technique of Southern blotting.
RESTRICTION ENDONUCLEASE ,TYPES , MECHANISM , APPLICATION