Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase".[1]
Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall in both categories. Well known nucleases are deoxyribonuclease and ribonuclease.
Introduction
In the late 1960s, scientists Stuart Linn and Werner Arber isolated examples of the two types of enzymes responsible for phage growth restriction in Escherichia coli (E. coli) bacteria.[2][3] 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.
Numerical Classification System
Most nucleases are classified by the Enzyme Commission number of the "Nomenclature Committee of the International Union of Biochemistry and Molecular Biology" as hydrolases (EC-number 3). The nucleases belong just like phosphodiesterase, lipase and phosphatase to the esterases (EC-number 3.1), a subgroup of the hydrolases. The esterases to which nucleases belong are classified with the EC-numbers 3.1.11 - EC-number 3.1.31.
Site-specific nuclease
Structure specific nuclease
For details see flap endonuclease.
Sequence specific nuclease
This important development came when 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. 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.
| Enzyme | Source | Recognition Sequence | Cut |
|---|---|---|---|
| HindII | Haemophilus influenzae |
5' GTYRAC 3' CARYTG |
5' GTY RAC 3' CAR YTG |
| R = A or G; Y = C or T | |||
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.
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. 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 | Escherichia coli | 3'CTTAAG |
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 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.
Meganucleases
The frequency at which a particular nuclease will cut a given DNA molecule depends on the complexity of the DNA and the length of the nuclease's recognition sequence; due to the statistical likelihood of finding the bases in a particular order by chance, a longer recognition sequence will result in less frequent digestion. For example, a given four-base sequence (corresponding to the recognition site for a hypothetical nuclease) would be predicted to occur every 256 base pairs on average (where 4^4=256), but any given six-base sequence would be expected to occur once every 4,096 base pairs on average (4^6=4096).
One unique family of nucleases is the meganucleases, which are characterized by having larger, and therefore less common, recognition sequences consisting of 12 to 40 base pairs. These nucleases are particularly useful for genetic engineering and Genome engineering applications in complex organisms such as plants and mammals, where typically larger genomes (numbering in the billions of base pairs) would result in frequent and deleterious site-specific digestion using traditional nucleases.
See also
References
- ↑ Avery, O.T., MacLeod, C.M., McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. J. Exp. Med. 79: 137-158.
- ↑ Linn S., Arber, W. (1968). Host specificity of DNA produced by Escherichia coli, X. In vitro restriction of phage fd replicative form. Proc. Natl. Acad. Sci. USA. 59:1300-1306
- ↑ Arber, W., Linn S. (1969) DNA modification and restriction. Annu. Rev. Biochem. 38:467-500
External links
- Examples of Restriction Enzymes Chart
- Restriction Enzyme Action of EcoRI
- Enzyme glossary
- Nucleases (Main source of the page...)