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    Restriction Endonuclease

    Introduction

    Restriction enzyme, also called restriction endonuclease, is a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infectious organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate DNA fragments, such as those containing genes; for this reason, they are indispensable tools of recombinant DNA technology (genetic engineering).

    A bacterium uses a restriction enzyme to defend itself against bacterial viruses called bacteriophages or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so it can replicate. The restriction enzyme prevents phage DNA from replicating by cutting it into many pieces. Restriction enzymes got their name from their ability to restrict or limit the number of bacteriophage strains that can infect a bacterium.

    Each restriction enzyme recognizes a specific short sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule: adenine, cytosine, thymine, and guanine). These regions are called recognition sequences or recognition sites and are randomly distributed throughout the DNA. Different bacterial species produce restriction enzymes that recognize different nucleotide sequences.

    When a restriction endonuclease recognizes a sequence, it cuts the DNA molecule by catalyzing hydrolysis (cleavage of a chemical bond by the addition of a water molecule) of the bond between adjacent nucleotides. The bacteria prevent their own DNA from being degraded in this way by disguising their recognition sequences.

    Enzymes called methylases to add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

    Restriction enzymes were discovered and characterized in the late 1960s and early 1970s by molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. The ability of enzymes to cut DNA at precise locations allowed researchers to isolate gene-containing fragments and recombine them with other DNA molecules, ie to clone genes.

    The names of restriction enzymes are derived from the genus, species, and strain designations of the bacteria that produce them; for example, the enzyme EcoRI is produced by the Escherichia coli strain RY13. Restriction enzymes are believed to have originated from a common ancestral protein and evolved to recognize specific sequences through processes such as genetic recombination and gene amplification.

    What is a restriction enzyme?

    Restriction enzymes are traditionally classified into four types based on subunit composition, cleavage position, sequence specificity, and cofactor requirements. However, amino acid sequencing has uncovered an extraordinary variety among restriction enzymes, revealing that, at the molecular level, there are many more than four different types.

    Types of restriction endonucleases

    Type I enzymes

    Type I enzymes are complex, multi-subunit restriction and modification enzymes that cut DNA randomly away from its recognition sequences. Originally thought to be rare, we now know from analysis of sequenced genomes that they are common. Type I enzymes are of considerable biochemical interest but are of little practical value as they do not produce discrete restriction fragments or distinct gel band patterns.

    Type II enzymes

    Type II enzymes cut DNA at defined positions near or within their recognition sequences. They produce discrete restriction fragments and distinct gel band patterns and are the predominant class used in the laboratory for routine DNA analysis and gene cloning. Instead of forming a single family of related proteins, type II enzymes are a collection of unrelated proteins of many different types.

    Type II enzymes often differ so completely in amino acid sequence from one another and, indeed, from any other known protein, that they exemplify the class of rapidly evolving proteins that are often indicative of involvement in host-infection interactions parasite.

    Type IIS Enzymes

    The next most common Type II enzymes generally referred to as “Type IIS” are those such as FokI (NEB #R0109) and AlwI (NEB #R0513) that cleave off their recognition sequence to one side. These enzymes are intermediate in size, 400 to 650 amino acids in length, and recognize sequences that are continuous and asymmetric.

    They comprise two distinct domains, one for DNA binding and the other for DNA cleavage. They are thought to bind DNA as monomers for the most part, but cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some type of IIS enzymes is much more active on DNA molecules that contain multiple recognition sites.

    Type IIG enzymes

    Type IIG restriction enzymes, the third major type of type II enzyme, are large combination restriction and modification enzymes, 850 to 1250 amino acids in length, in which the two enzyme activities reside on the same protein chain.

    These enzymes cleave outside of their recognition sequences and can be classified as those that recognize continuous sequences (eg, AcuI (NEB #R0641): CTGAAG) and cleave on one side only; and those that recognize discontinuous sequences (eg, BcgI (NEB #R0545): CGANNNNNNTGC) and cleave on both sides releasing a small fragment containing the recognition sequence.

    The amino acid sequences of these enzymes are varied, but their organization is consistent. They comprise an N-terminal DNA cleavage domain linked to a DNA modification domain and one or two DNA sequence-specificity domains that form the C-terminus or occur as a separate subunit. When these enzymes bind to their substrates, they switch to restriction mode to cleave DNA or to modification mode to methylate it.

    Type III enzymes

    Type III enzymes are also large combination modification and restriction enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to achieve cleavage; they rarely give full summaries.

    Type IV enzymes

    Type IV enzymes recognize modified DNA, typically methylated, and are exemplified by the E. coli McrBC and Mrr systems.