thumb|Depiction of the [[restriction enzyme (endonuclease) HindIII cleaving a double-stranded DNA molecule at a valid restriction site ().]]

In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids. Nucleases variously affect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

There are two primary classifications based on the locus of activity. Exonucleases digest nucleic acids from the ends. Endonucleases act on regions in the middle of target molecules. They are further subcategorized as deoxyribonucleases and ribonucleases. The former acts on DNA, the latter on RNA. 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.

An important development came when H.O. Smith, K.W. Wilcox, and T.J. Kelly, 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. They found that the HindII enzyme always cuts directly in the center of this sequence (between the 3rd and 4th base pairs).

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.

Structure

Nuclease primary structure is by and large poorly conserved and minimally conserved at active sites, the surfaces of which primarily comprise acidic and basic amino acid residues. Nucleases can be classified into folding families.]]

Site recognition

A nuclease must associate with a nucleic acid before it can cleave the molecule. That entails a degree of recognition. Nucleases variously employ both nonspecific and specific associations in their modes of recognition and binding. Both modes play important roles in living organisms, especially in DNA repair.

Staggered cutting

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 recognition sequence <code>5'—GAATTC—3'</code>.

{| class="wikitable floatright"

!Enzyme!!Source!!Recognition Sequence!!Cut

|-

|HindIII

|Haemophilus influenzae

|

<div class="center">5'–AAGCTT–3'<br />

3'–TTCGAA–5'</div>

|

<div class="center">5'– –3'<br />

3'– –5'</div>

|-

|EcoRI

|Escherichia coli

| <div class="center">5'–GAATTC-3'<br />

3'–CTTAAG–5'</div>

| <div class="center">5'– –3'<br />

3'– –5'</div>

|-

|BamHI

|Bacillus amyloliquefaciens

| <div class="center">5'–GGATCC–3'<br />

3'–CCTAGG–5'</div>

| <div class="center">5'– –3'<br />

3'– –5'</div>

|}

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&mdash;both 3' and 5'&mdash;are sometimes called "sticky ends" because they tend to bond with complementary sequences of bases. In other words, if an unpaired length of bases <code>5'—AATT—3'</code> encounters another unpaired length with the sequence <code>3'—TTAA—5'</code> they will bond to each other&mdash;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.

Role in nature

DNA repair

With all cells depending on DNA as the medium of genetic information, genetic quality control is an essential function of all organisms. DNA replication is an error prone process, and DNA molecules themselves are vulnerable to modification by many metabolic and environmental stressors. Ubiquitous examples include reactive oxygen species, near ultraviolet, and ionizing radiation. Many nucleases participate in DNA repair by recognizing damage sites and cleaving them from the surrounding DNA. These enzymes function independently or in complexes. Most nucleases involved in DNA repair are not sequence-specific. They recognize damage sites through deformation of double stranded DNA (dsDNA) secondary structure.

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

  • HindIII
  • Ligase
  • Micrococcal nuclease
  • Nuclease protection assay
  • P1 nuclease
  • PIN domain
  • Polymerase
  • Serratia marcescens nuclease (benzonase)
  • S1 nuclease

References

  • Examples of Restriction Enzymes Chart
  • Restriction Enzyme Action of EcoRI
  • Enzyme glossary
  • Nucleases (Main source of the page...)