Nuclease
Encyclopedia
A nuclease is an enzyme
capable of cleaving the phosphodiester bond
s between the nucleotide subunits of nucleic acid
s. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase".
Nucleases are usually further divided into endonuclease
s and exonuclease
s, although some of the enzymes may fall in both categories.
isolated examples of the two types of enzymes responsible for phage growth restriction in Escherichia coli
(E. coli) bacteria. One of these enzymes added a
methyl group
to the DNA, generating methylated DNA
, while the other cleaved unmethylated DNA at a wide variety of locations along the length of the molecule. The first type of enzyme was called a "methylase
" and the other a "restriction nuclease". These enzymatic tools were important to scientists who were gathering the tools needed to "cut and paste
" DNA molecules. What was then needed was a tool that would cut DNA at specific sites, rather than at random sites along the length of the molecule, so that scientists could cut DNA
molecules in a predictable and reproducible way.
in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide
sequence. Working with Haemophilus influenzae
bacteria, this group isolated an enzyme, called HindII
, that always cut DNA molecules at a particular point within a specific sequence of six base pairs.
They found that the HindII enzyme always cuts directly in the center of this sequence. Wherever this particular sequence of six base pairs occurs unmodified in a DNA molecule, HindII will cleave both DNA strands between the 3rd and 4th base pairs of the sequence. Moreover, HindII will only cleave a DNA molecule at this particular site. For this reason, this specific base sequence is known as the "recognition sequence
" for HindII.
HindII is only one example of the class of enzymes known as restriction nucleases. In fact, more than 900 restriction enzymes, some sequence specific and some not, have been isolated from over 230 strains of bacteria since the initial discovery of HindII. These restriction enzymes generally have names that reflect their origin—The first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated. For example EcoRI
comes from Escherichia coli
RY13 bacteria, while HindII comes from Haemophilus influenzae
strain Rd. Numbers following the nuclease names indicate the order in which the enzymes were isolated from single strains of bacteria: EcoRI
, EcoRII. Nucleases are further described by addition of the prefix "endo" or "exo" to the name: The term "endonuclease
" applies to nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule. A nuclease that functions by removing nucleotides from the ends of the DNA molecule is called an exonuclease
.
When the enzyme encounters this sequence, it cleaves each backbone between the G and the closest A base residues. Once the cuts have been made, the resulting fragments are held together only by the relatively weak hydrogen bonds that hold the complementary bases to each other. The weakness of these bonds allows the DNA fragments to separate from each other. Each resulting fragment has a protruding 5' end composed of unpaired bases. Other enzymes create cuts in the DNA backbone which result in protruding 3' ends. Protruding ends—both 3' and 5'—are sometimes called "sticky ends" because they tend to bond with complementary sequences of bases. In other words, if an unpaired length of bases (5' A A T T 3') encounters another unpaired length with the sequence (3' T T A A 5') they will bond to each other—they are "sticky" for each other. Ligase
enzyme is then used to join the phosphate backbones of the two molecules. The cellular origin, or even the species origin, of the sticky ends does not affect their stickiness. Any pair of complementary sequences will tend to bond, even if one of the sequences comes from a length of human DNA, and the other comes from a length of bacterial DNA. In fact, it is this quality of stickiness that allows production of recombinant DNA molecules, molecules which are composed of DNA from different sources, and which has given birth to the genetic engineering
technology.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
capable of cleaving the phosphodiester bond
Phosphodiester bond
A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. Phosphodiester bonds are central to all known life, as they make up the backbone of each helical strand of DNA...
s between the nucleotide subunits of nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase".
Nucleases are usually further divided into endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
s and exonuclease
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
s, although some of the enzymes may fall in both categories.
Introduction
In the late 1960s, scientists Stuart Linn and Werner ArberWerner Arber
Werner Arber is a Swiss microbiologist and geneticist. Along with American researchers Hamilton Smith and Daniel Nathans, Werner Arber shared the 1978 Nobel Prize in Physiology or Medicine for the discovery of restriction endonucleases...
isolated examples of the two types of enzymes responsible for phage growth restriction in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
(E. coli) bacteria. One of these enzymes added a
methyl group
Methyl group
Methyl group is a functional group derived from methane, containing one carbon atom bonded to three hydrogen atoms —CH3. The group is often abbreviated Me. Such hydrocarbon groups occur in many organic compounds. The methyl group can be found in three forms: anion, cation and radical. The anion...
to the DNA, generating methylated DNA
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
, while the other cleaved unmethylated DNA at a wide variety of locations along the length of the molecule. The first type of enzyme was called 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...
" and the other a "restriction nuclease". These enzymatic tools were important to scientists who were gathering the tools needed to "cut and paste
Cut and paste
In human-computer interaction, cut and paste and copy and paste offer user-interface interaction techniques for transferring text, data, files or objects from a source to a destination. Most ubiquitously, users require the ability to cut and paste sections of plain text...
" DNA molecules. What was then needed was a tool that would cut DNA at specific sites, rather than at random sites along the length of the molecule, so that scientists could cut 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...
molecules in a predictable and reproducible way.
Sequence specific nuclease
This important development came when H.O. Smith, K.W. Wilcox, and T.J. Kelley, working at Johns Hopkins UniversityJohns Hopkins University
The Johns Hopkins University, commonly referred to as Johns Hopkins, JHU, or simply Hopkins, is a private research university based in Baltimore, Maryland, United States...
in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
sequence. Working with Haemophilus influenzae
Haemophilus influenzae
Haemophilus influenzae, formerly called Pfeiffer's bacillus or Bacillus influenzae, Gram-negative, rod-shaped bacterium first described in 1892 by Richard Pfeiffer during an influenza pandemic. A member of the Pasteurellaceae family, it is generally aerobic, but can grow as a facultative anaerobe. H...
bacteria, this group isolated an enzyme, called HindII
HindII
H.O. Smith, K.W. Wilcox, and T.J. Kelley, working at Johns Hopkins University in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence...
, that always cut DNA molecules at a particular point within a specific sequence of six base pairs.
| Enzyme | Source | Recognition Sequence | Cut |
|---|---|---|---|
| HindII HindII H.O. Smith, K.W. Wilcox, and T.J. Kelley, working at Johns Hopkins University in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence... |
Haemophilus influenzae Haemophilus influenzae Haemophilus influenzae, formerly called Pfeiffer's bacillus or Bacillus influenzae, Gram-negative, rod-shaped bacterium first described in 1892 by Richard Pfeiffer during an influenza pandemic. A member of the Pasteurellaceae family, it is generally aerobic, but can grow as a facultative anaerobe. H... |
5' GTYRAC 3' CARYTG |
5' GTY RAC 3' CAR YTG |
| R = A Adenine Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA... or G Guanine Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with... ; Y = C Cytosine Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine... or T Thymine Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at... |
|||
They found that the HindII enzyme always cuts directly in the center of this sequence. Wherever this particular sequence of six base pairs occurs unmodified in a DNA molecule, HindII will cleave both DNA strands between the 3rd and 4th base pairs of the sequence. Moreover, HindII will only cleave a DNA molecule at this particular site. For this reason, this specific base sequence is known as the "recognition sequence
Recognition sequence
The recognition sequence, sometimes also referred to as recognition site, of any DNA-binding protein motif that exhibits binding specificity, refers to the DNA sequence , to which the domain is specific...
" for HindII.
HindII is only one example of the class of enzymes known as restriction nucleases. In fact, more than 900 restriction enzymes, some sequence specific and some not, have been isolated from over 230 strains of bacteria since the initial discovery of HindII. These restriction enzymes generally have names that reflect their origin—The first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated. For example EcoRI
EcoRI
EcoRI is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.In molecular biology it is used as a restriction enzyme. It creates sticky ends with 5' end overhangs...
comes from Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
RY13 bacteria, while HindII comes from Haemophilus influenzae
Haemophilus influenzae
Haemophilus influenzae, formerly called Pfeiffer's bacillus or Bacillus influenzae, Gram-negative, rod-shaped bacterium first described in 1892 by Richard Pfeiffer during an influenza pandemic. A member of the Pasteurellaceae family, it is generally aerobic, but can grow as a facultative anaerobe. H...
strain Rd. Numbers following the nuclease names indicate the order in which the enzymes were isolated from single strains of bacteria: EcoRI
EcoRI
EcoRI is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.In molecular biology it is used as a restriction enzyme. It creates sticky ends with 5' end overhangs...
, EcoRII. Nucleases are further described by addition of the prefix "endo" or "exo" to the name: The term "endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
" applies to nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule. A nuclease that functions by removing nucleotides from the ends of the DNA molecule is called an exonuclease
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
.
Endonucleases and DNA fragments
A restriction endonuclease functions by "scanning" the length of a DNA molecule. Once it encounters its particular specific recognition sequence, it will bind to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones. The positions of these two cuts, both in relation to each other, and to the recognition sequence itself, are determined by the identity of the restriction endonuclease used to cleave the molecule in the first place. Different endonucleases yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way, no matter what DNA molecule it is acting on. Once the cuts have been made, the DNA molecule will break into fragments.Endonucleases and sticky ends
Not all restriction endonucleases cut symmetrically and leave blunt ends like HindII described above. Many endonucleases cleave the DNA backbones in positions that are not directly opposite each other, creating overhangs. For example, the nuclease EcoRI has the following recognition sequence:| Enzyme | Source | Recognition Sequence | Cut |
|---|---|---|---|
| EcoRI EcoRI EcoRI is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.In molecular biology it is used as a restriction enzyme. It creates sticky ends with 5' end overhangs... |
Escherichia coli Escherichia coli Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls... |
5'GAATTC 3'CTTAAG |
5'---G AATTC---3' 3'---CTTAA G---5' |
When the enzyme encounters this sequence, it cleaves each backbone between the G and the closest A base residues. Once the cuts have been made, the resulting fragments are held together only by the relatively weak hydrogen bonds that hold the complementary bases to each other. The weakness of these bonds allows the DNA fragments to separate from each other. Each resulting fragment has a protruding 5' end composed of unpaired bases. Other enzymes create cuts in the DNA backbone which result in protruding 3' ends. Protruding ends—both 3' and 5'—are sometimes called "sticky ends" because they tend to bond with complementary sequences of bases. In other words, if an unpaired length of bases (5' A A T T 3') encounters another unpaired length with the sequence (3' T T A A 5') they will bond to each other—they are "sticky" for each other. Ligase
Ligase
In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...
enzyme is then used to join the phosphate backbones of the two molecules. The cellular origin, or even the species origin, of the sticky ends does not affect their stickiness. Any pair of complementary sequences will tend to bond, even if one of the sequences comes from a length of human DNA, and the other comes from a length of bacterial DNA. In fact, it is this quality of stickiness that allows production of recombinant DNA molecules, molecules which are composed of DNA from different sources, and which has given birth to the genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
technology.
See also
- Nuclease protection assayNuclease protection assayNuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration...
- Micrococcal nucleaseMicrococcal nucleaseMicrococcal Nuclease is an endo-exonuclease that preferentially digests single-stranded nucleic acids.The rate of cleavage is 30 times greater at the 5' side of A or T than at G or C and results in the production of mononucleotides and oligonucleotides with terminal 3'-phosphates...
- S1 nucleaseS1 nucleaseS1 nuclease is an endonuclease that is active against single-stranded DNA and RNA molecules. It is five times more active on DNA than RNA. Its reaction products are oligonucleotides or single nucleotides with 5' phosphoryl groups...
- P1 nucleaseP1 nucleaseP1 nuclease is a nuclease that works on single-stranded DNA as well as RNA. P1 nuclease cleaves at every position yielding nucleoside 5' monophosphates. It is useful at removing single stranded strands hanging off the end of double stranded DNA and at completely cleaving melted DNA for simple DNA...
- HindIIIHindIIIHindIII is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the palindromic DNA sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis....
- PIN domainPIN domainIn molecular biology the PIN domain is a protein domain that is about 130 amino acids in length. The PIN domain was named after its identification in the N-terminus of the PilT protein . PIN domains function as nuclease enzymes that cleave single stranded RNA in a sequence dependent manner.PIN...