Restriction modification system
Encyclopedia
The restriction modification system (RM system) is used by bacteria
, and perhaps other prokaryotic
organisms to protect themselves from foreign DNA
, such as the one borne by bacteriophage
s. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit (restrict) the growth of viruses grown in previous strains. This effect was attributed to sequence-specific restriction enzyme
s.
Bacteria have restriction enzyme
s, also called restriction endonucleases, which cleave double stranded DNA
at specific points into fragments, which are then degraded further by other endonucleases. This prevents infection by effectively destroying the foreign DNA
introduced by an infectious agent (such as a bacteriophage
). Approximately one quarter of known bacteria possess RM systems and of those about one half have more than one type of system.
Given that the sequences that the restriction enzymes recognize are very short, the bacterium itself will almost certainly have many of these sequences present in its own DNA. Therefore, in order to prevent destruction of its own DNA by the restriction enzymes, the bacterium marks its own DNA by adding methyl groups to it. This modification must not interfere with the DNA base-pairing, and therefore, usually only a few specific bases are modified on each strand.
Endonucleases cleave internal/non-terminal phosphodiester bonds. Restriction endonucleases cleave internal phosphodiester bonds only after recognising specific sequences in DNA which are usually 4-6 base pairs long, and often palindromic.
activity and a methylase
activity. They were named in the order of discovery, although the type II system is the most common.
Type I systems are the most complex, consisting of three polypeptides: R (restriction), M (modification), and S (specificity). The resulting complex can both cleave and methylate DNA. Both reactions require ATP, and cleavage often occurs a considerable distance from the recognition site. The S subunit determines the specificity of both restriction and methylation. Cleavage occurs at variable distances from the recognition sequence, so discrete bands are not easily visualized by gel electrophoresis
.
Type II systems are the simplest and the most prevalent. Instead of working as a complex, the methyltransferase and endonuclease are encoded as two separate proteins and act independently (there is no specificity protein). Both proteins recognize the same recognition site, and therefore compete for activity. The methyltransferase acts as a monomer
, methylating the duplex one strand at a time. The endonuclease acts as a homodimer
, which facilitates the cleavage of both strands. Cleavage occurs at a defined position close to or within the recognition sequence, thus producing discrete fragments during gel electrophoresis. For this reason, Type II systems are used in labs for DNA analysis
and gene cloning
.
Type III systems have R and M proteins that form a complex of modification and cleavage. The M protein, however, can methylate on its own. Methylation also only occurs on one strand of the DNA unlike most other known mechanisms. The heterodimer
formed by the R and M proteins competes with itself by modifying and restricting the same reaction. This results in incomplete digestion.
s and selected because of the resistance provided by the methylation enzyme. Once the plasmid begins to replicate, the methylation enzyme will be produced and methylate the plasmid DNA, protecting it from a specific restriction enzyme.
Some viruses have evolved ways of subverting the restriction modification system, usually by modifying their own DNA, by adding methyl or glycosyl
groups to it, thus blocking the restriction enzymes. Other viruses, such as bacteriophages T3 and T7, encode proteins that inhibit the restriction enzymes.
To counteract these viruses, some bacteria have evolved restriction systems which only recognize and cleave modified DNA, but do not act upon the host's unmodified DNA. Some prokaryotes have developed multiple types of restriction modification systems.
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, and perhaps other prokaryotic
Prokaryote
The prokaryotes are a group of organisms that lack a cell nucleus , or any other membrane-bound organelles. The organisms that have a cell nucleus are called eukaryotes. Most prokaryotes are unicellular, but a few such as myxobacteria have multicellular stages in their life cycles...
organisms to protect themselves from foreign DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, such as the one borne by bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
s. This phenomenon was first noticed in the 1950s. Certain bacteria strains were found to inhibit (restrict) the growth of viruses grown in previous strains. This effect was attributed to sequence-specific restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s.
Bacteria have restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s, also called restriction endonucleases, which cleave double stranded DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
at specific points into fragments, which are then degraded further by other endonucleases. This prevents infection by effectively destroying the foreign DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
introduced by an infectious agent (such as a bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
). Approximately one quarter of known bacteria possess RM systems and of those about one half have more than one type of system.
Given that the sequences that the restriction enzymes recognize are very short, the bacterium itself will almost certainly have many of these sequences present in its own DNA. Therefore, in order to prevent destruction of its own DNA by the restriction enzymes, the bacterium marks its own DNA by adding methyl groups to it. This modification must not interfere with the DNA base-pairing, and therefore, usually only a few specific bases are modified on each strand.
Endonucleases cleave internal/non-terminal phosphodiester bonds. Restriction endonucleases cleave internal phosphodiester bonds only after recognising specific sequences in DNA which are usually 4-6 base pairs long, and often palindromic.
Types of restriction modification system
There are five kinds of restriction modification system: type I, type II, type IIS, type III and type IV, all with restriction enzymeRestriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
activity and a methylase
Methylase
A methylase is an enzyme that attaches a methyl group to a molecule.These are found in prokaryotes and eukaryotes. Bacteria use methylase to differentiate between foreign genetic material and their own, thus protecting their DNA from their own immune system...
activity. They were named in the order of discovery, although the type II system is the most common.
Type I systems are the most complex, consisting of three polypeptides: R (restriction), M (modification), and S (specificity). The resulting complex can both cleave and methylate DNA. Both reactions require ATP, and cleavage often occurs a considerable distance from the recognition site. The S subunit determines the specificity of both restriction and methylation. Cleavage occurs at variable distances from the recognition sequence, so discrete bands are not easily visualized by gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
.
Type II systems are the simplest and the most prevalent. Instead of working as a complex, the methyltransferase and endonuclease are encoded as two separate proteins and act independently (there is no specificity protein). Both proteins recognize the same recognition site, and therefore compete for activity. The methyltransferase acts as a monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
, methylating the duplex one strand at a time. The endonuclease acts as a homodimer
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
, which facilitates the cleavage of both strands. Cleavage occurs at a defined position close to or within the recognition sequence, thus producing discrete fragments during gel electrophoresis. For this reason, Type II systems are used in labs for DNA analysis
Genetic fingerprinting
DNA profiling is a technique employed by forensic scientists to assist in the identification of individuals by their respective DNA profiles. DNA profiles are encrypted sets of numbers that reflect a person's DNA makeup, which can also be used as the person's identifier...
and gene cloning
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
.
Type III systems have R and M proteins that form a complex of modification and cleavage. The M protein, however, can methylate on its own. Methylation also only occurs on one strand of the DNA unlike most other known mechanisms. The heterodimer
Protein dimer
In biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
formed by the R and M proteins competes with itself by modifying and restricting the same reaction. This results in incomplete digestion.
Uses
RM systems can be cloned into plasmidPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s and selected because of the resistance provided by the methylation enzyme. Once the plasmid begins to replicate, the methylation enzyme will be produced and methylate the plasmid DNA, protecting it from a specific restriction enzyme.
Some viruses have evolved ways of subverting the restriction modification system, usually by modifying their own DNA, by adding methyl or glycosyl
Glycosyl
A glycosyl group is a univalent free radical or substituent structure obtained by removing the hemiacetal hydroxyl group from the cyclic form of a monosaccharide and, by extension, of a lower oligosaccharide....
groups to it, thus blocking the restriction enzymes. Other viruses, such as bacteriophages T3 and T7, encode proteins that inhibit the restriction enzymes.
To counteract these viruses, some bacteria have evolved restriction systems which only recognize and cleave modified DNA, but do not act upon the host's unmodified DNA. Some prokaryotes have developed multiple types of restriction modification systems.