Restriction Enzyme (Restriction Endonuclease)

A restriction enzyme (also called a restriction endonuclease) is a type of protein that can cut DNA molecules at specific sequences of nucleotides. These sequences are called restriction sites and they are usually four to eight base pairs long and palindromic, meaning they read the same on both strands of the DNA.

Restriction enzymes are produced by bacteria as a defense mechanism against foreign DNA, such as from viruses or other bacteria. By cutting the foreign DNA into smaller fragments, the bacteria can prevent it from replicating or expressing harmful genes. The bacteria protect their own DNA from being cut by the same restriction enzymes by adding methyl groups to their restriction sites, making them unrecognizable by the enzymes.

Restriction enzymes are widely used in molecular biology and biotechnology as tools for manipulating DNA. They can be used to cut DNA into smaller pieces for analysis, to insert or remove genes from DNA molecules, to create recombinant DNA by joining fragments from different sources, and to generate DNA libraries for sequencing or cloning. Restriction enzymes are also essential for some techniques such as polymerase chain reaction (PCR), gel electrophoresis, Southern blotting, and DNA fingerprinting.

Restriction enzymes are proteins that can cut DNA molecules at specific sequences, called restriction sites. These enzymes are found in bacteria and archaea, where they act as a defense mechanism against invading viruses.

Restriction enzymes recognize and bind to their restriction sites with high specificity and accuracy. Depending on the type of enzyme, the cleavage can occur within or near the restriction site, and produce different types of ends on the DNA fragments.

Some restriction enzymes cut the DNA straight across the double helix, producing blunt ends with no overhangs. For example, EcoRV cuts at the sequence GAT|ATC, leaving blunt ends like this:

5`-GATATC-3`
    |
3`-CTATAG-5`

Other restriction enzymes make staggered cuts in the DNA, leaving single-stranded overhangs on either end. These overhangs are complementary to each other and can base pair with other DNA fragments with matching ends. For this reason, they are called sticky ends. For example, EcoRI cuts at the sequence G|AATTC, leaving sticky ends like this:

5`-GAATTC-3`
  |
3`-CTTAA G-5`

The length and sequence of the overhangs vary depending on the restriction enzyme. Some enzymes produce 5` overhangs (as shown above), while others produce 3` overhangs (such as KpnI, which cuts at GGTAC|C). Some enzymes recognize palindromic sequences (such as EcoRI), while others recognize asymmetric sequences (such as BsaI, which cuts at GGTCTC N1/N5).

The ability of restriction enzymes to cut DNA into specific fragments is very useful for molecular cloning and biotechnology applications. By using the same or compatible restriction enzymes, DNA fragments from different sources can be joined together by complementary base pairing and then sealed by another enzyme called DNA ligase. This allows researchers to insert genes or other pieces of DNA into plasmids or other vectors for further manipulation or expression.

The natural source of restriction enzymes 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. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

Restriction enzymes are found in bacteria (and other prokaryotes). They recognize and bind to specific sequences of DNA, called restriction sites. Each restriction enzyme recognizes just one or a few restriction sites. When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule. Typically, the cut is at or near the restriction site and occurs in a tidy, predictable pattern.

Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methylases add methyl groups (—CH 3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease.

Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. To be able to sequence DNA, it is first necessary to cut it into smaller fragments.

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).

Restriction enzymes are proteins that recognize and cut specific DNA sequences, called restriction sites. Restriction sites are usually 4 to 8 base pairs long and have a palindromic structure, meaning that they read the same on both strands of DNA when oriented in the same direction. For example, the restriction site for EcoRI is GAATTC, which is the same as CTTAAG when read from 5` to 3` on both strands.

Restriction enzymes can cut DNA at or near their recognition sites, depending on the type of enzyme. Some enzymes cut within the recognition site, while others cut at a fixed distance away from it. The cut can be either straight across the DNA molecule, producing blunt ends, or staggered, producing sticky ends with single-stranded overhangs. For instance, EcoRI cuts between G and A on both strands, leaving sticky ends with a 4-base overhang:

5` ...G AATTC... 3`
3` ...CTTAA G... 5`

Sticky ends are useful for DNA cloning because they can anneal with complementary overhangs from other DNA fragments that have been cut by the same enzyme. This allows the fragments to be joined together by DNA ligase, which seals the gaps between them.

Different restriction enzymes have different recognition sites and cutting patterns. For example, HindIII recognizes AAGCTT and cuts between A and A on both strands, leaving sticky ends with a 5-base overhang:

5` ...A AGCTT... 3`
3` ...TTCGA A... 5`

EcoRV recognizes GATATC and cuts in the middle of the site, leaving blunt ends:

5` ...GAT ATC... 3`
3` ...CTA TAG... 5`

PvuII recognizes CAGCTG and cuts between C and T on both strands, leaving blunt ends:

5` ...CA GCTG... 3`
3` ...GT CGAC... 5`

Restriction enzymes are named after the bacteria from which they are isolated, using a system based on bacterial genus, species and strain. For example, EcoRI comes from Escherichia coli strain RY13, HindIII comes from Haemophilus influenzae strain Rd, and PvuII comes from Proteus vulgaris strain II.

Restriction enzymes are widely used in molecular biology for DNA manipulation and analysis. They can be used to cut DNA into fragments of different sizes and origins, which can then be separated by gel electrophoresis, cloned into plasmids or other vectors, or sequenced.

There are hundreds of restriction enzymes available commercially, each with its own recognition site and cutting pattern. Some examples of restriction enzymes and their recognition sites are shown in the table below:

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.

The mechanism of cleavage by restriction enzymes depends on their type and specificity. Some enzymes cut straight across the molecule at the symmetrical axis producing blunt ends, while others cut between the same two bases away from the point of symmetry on two strands, thus, producing a staggering break with sticky or protruding ends. The sticky ends can be used to join DNA fragments from different sources by complementary base pairing.

Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. They require both ATP and S-adenosyl-L-methionine to function and are multifunctional proteins with both restriction and methylase activities. They can be blocked by positive supercoiling or a Holliday junction, which trigger DNA cleavage.

Type II restriction enzymes cleave within or at short specific distances from a recognition site. They are the most widely used in molecular biology because they have well-defined recognition sequences and cleavage sites. They usually require magnesium as a cofactor and are single function (restriction) enzymes independent of methylase.

Type III restriction enzymes cleave at sites a short distance from a recognition site. They require ATP (but do not hydrolyze it) and S-adenosyl-L-methionine stimulates the reaction but is not required. They exist as part of a complex with a modification methylase.

Type IV restriction enzymes target modified DNA, such as methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA. They have diverse specificities and mechanisms of action.

Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors. Here is a brief overview of each type:

• Type I restriction enzymes are also called restriction endonucleases. They are made of two long strands of DNA joined together. These restriction enzymes recognize certain sequences of DNA and cleave them at a site that is random and far from the recognition site. They require both ATP and S-adenosyl-L-methionine to function and have both restriction and methylase activities.
• Type II restriction enzymes are the most widely used in biotechnology and molecular biology. They are simpler and smaller than type I enzymes and consist of single proteins. They recognize specific sequences of DNA and cleave them within or at short specific distances from the recognition site. Most of them require magnesium as a cofactor and have only restriction activity independent of methylase. They produce either sticky ends or blunt ends depending on how they cut the DNA.
• Type III restriction enzymes are similar to type I enzymes in that they recognize specific sequences of DNA and cleave them at sites that are distant from the recognition site. However, they cut the DNA only when two copies of the recognition sequence are present on opposite strands of the same DNA molecule. They require ATP (but do not hydrolyze it) and S-adenosyl-L-methionine stimulates the reaction but is not required. They exist as part of a complex with a modification methylase.
• Type IV restriction enzymes are less common and less well characterized than the other types. They target modified DNA, such as methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA. They may have different mechanisms of action and recognition patterns.

Among these types, type II restriction enzymes are the most useful for DNA cloning because they produce fragments with defined ends that can be easily ligated together.

Restriction enzymes are named according to a standard convention that reflects their origin and characteristics. The name consists of three or four letters followed by a Roman numeral. The letters are derived from the genus, species and strain of the bacterial source, while the numeral indicates the order of discovery. For example, EcoRI is a restriction enzyme isolated from Escherichia coli strain RY13, and it was the first enzyme of this type identified in this bacterium.

The first letter of the enzyme name is capitalized, while the rest are lowercase. The Roman numeral is written after a space or a dash. Sometimes, an additional letter is added to the name to indicate the subtype or specificity of the enzyme. For example, HpaII is a restriction enzyme isolated from Haemophilus parainfluenzae, and it recognizes the same sequence as MspI, which is isolated from Moraxella sp. However, HpaII only cleaves DNA that is unmethylated at the recognition site, while MspI cleaves both methylated and unmethylated DNA.

The recognition sequences of restriction enzymes are usually palindromic, meaning that they read the same on both strands of DNA when oriented in the same direction. The recognition sequences vary in length from 4 to 8 base pairs (bp), and some enzymes have degenerate or ambiguous sequences that can tolerate variations at certain positions. The recognition sequences are written in the 5` to 3` direction, using the standard abbreviations for the four nucleotides: A, T, G and C. For example, the recognition sequence of EcoRI is 5`-GAATTC-3`, and that of HpaII is 5`-CCGG-3`.

Some restriction enzymes make staggered cuts in the DNA molecule, leaving short single-stranded overhangs at the ends of the fragments. These overhangs are called sticky or cohesive ends, because they can anneal with complementary sequences on other DNA molecules. Other restriction enzymes make blunt cuts in the DNA molecule, leaving no overhangs at the ends of the fragments. These ends are called blunt or flush ends, because they cannot anneal with other DNA molecules. The type of ends produced by a restriction enzyme depends on the position and orientation of its cleavage site relative to its recognition site. For example, EcoRI makes staggered cuts between G and A on both strands, leaving 4-bp overhangs with 5`-AATT-3` sequences. On the other hand, HpaII makes blunt cuts between C and G on both strands, leaving no overhangs.

The nomenclature of restriction enzymes is useful for identifying their source, sequence specificity and cleavage pattern. It also facilitates the comparison and selection of different enzymes for various applications in molecular biology and biotechnology.

Restriction enzymes are widely used in molecular biology and biotechnology for various purposes, such as:

• Recombinant DNA technology: Restriction enzymes can cut DNA molecules at specific sites and generate fragments with sticky or blunt ends. These fragments can be joined with other DNA molecules from different sources using DNA ligase, creating recombinant DNA molecules. For example, restriction enzymes can be used to insert a gene of interest into a plasmid vector for cloning or expression in bacteria.
• DNA fingerprinting: Restriction enzymes can be used to analyze the genetic variation among individuals or populations based on the patterns of DNA fragments generated by cutting their DNA samples with the same restriction enzyme. This technique is called restriction fragment length polymorphism (RFLP) and can be used for forensic identification, paternity testing, genetic diagnosis, and evolutionary studies.
• Gene mapping: Restriction enzymes can be used to locate the position of a gene on a chromosome by cutting the DNA into fragments and analyzing their sizes and distribution. This technique is called restriction mapping and can be used to construct physical maps of genomes or to identify the location of mutations or disease-causing genes.
• Gene editing: Restriction enzymes can be used to introduce specific changes in the DNA sequence by cutting the target site and replacing it with a new DNA fragment. This technique is called gene targeting and can be used to create transgenic organisms or to correct genetic defects. Alternatively, restriction enzymes can be used to create double-strand breaks in the DNA that trigger the cell`s own repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). This technique is called genome editing and can be used to modify or regulate gene expression or function.
• Molecular cloning: Restriction enzymes can be used to isolate and amplify a specific DNA fragment by cutting it out from the source DNA and inserting it into a suitable vector that can replicate in a host cell. This technique is called molecular cloning and can be used to produce large amounts of a desired DNA fragment or its encoded protein.
• Polymerase chain reaction (PCR): Restriction enzymes can be used to modify the ends of PCR primers or products by adding or removing restriction sites. This can facilitate the cloning, sequencing, or analysis of PCR products. For example, restriction enzymes can be used to create compatible ends for directional cloning or to introduce mutations or tags into PCR products.

Restriction enzymes are classified into different types based on their structure, cleavage site, specificity and cofactors. There are hundreds of restriction enzymes that have been isolated from various bacteria and are commercially available. Each enzyme has a unique name that indicates its bacterial origin and recognition sequence. Here are some examples of restriction enzymes from different types:

• EcoRI is a type II restriction enzyme that recognizes the sequence 5`-GAATTC-3` and cuts between G and A on both strands, producing sticky ends with 5` overhangs. It is derived from Escherichia coli strain RY13.
• BamHI is another type II restriction enzyme that recognizes the sequence 5`-GGATCC-3` and cuts between G and G on both strands, producing sticky ends with 5` overhangs. It is derived from Bacillus amyloliquefaciens strain H.
• DpnI is a type IV restriction enzyme that recognizes the sequence 5`-GATC-3` and cuts between G and A on both strands, but only if the adenine is methylated. It is derived from Diplococcus pneumoniae.
• HindIII is a type II restriction enzyme that recognizes the sequence 5`-AAGCTT-3` and cuts between A and A on both strands, producing sticky ends with 5` overhangs. It is derived from Haemophilus influenzae strain Rd.
• NotI is a type II restriction enzyme that recognizes the sequence 5`-GCGGCCGC-3` and cuts between G and C on both strands, producing sticky ends with 4-base 5` overhangs. It is derived from Nocardia otitidis-caviarum.
• SmaI is a type II restriction enzyme that recognizes the sequence 5`-CCCGGG-3` and cuts between C and G on both strands, producing blunt ends. It is derived from Serratia marcescens.

These are just some examples of restriction enzymes that are commonly used in molecular biology. Different restriction enzymes have different applications depending on their recognition sequences, cleavage patterns and compatibility with other enzymes. Restriction enzymes are essential tools for DNA cloning, mapping, sequencing, analysis and modification.

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