Nuclease

In

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.^[2]

History

In the late 1960s, scientists

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.^[5]

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.^[7]

Nonspecific endonucleases involved in DNA repair can scan DNA for

A site-specific nuclease forms far stronger associations by contrast. It draws DNA into the deep groove of its DNA-binding domain. This results in significant deformation of the DNA tertiary structure and is accomplished with a surfaces rich in basic (positively charged) residues. It engages in extensive electrostatic interaction with the DNA.^[7]

Some nucleases involved in DNA repair exhibit partial sequence-specificity. However most are nonspecific, instead recognizing structural abnormalities produced in the DNA

For details see flap endonuclease.

Sequence specific nuclease

There are 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,

Endonucleases

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.

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 5'—GAATTC—3'.

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 "

DNA mismatch repair in any given organism is effected by a suite of mismatch-specific endonucleases. In prokaryotes, this role is primarily filled by MutSLH and very short patch repair (VSP repair) associated proteins.

The MutSLH system (comprising

Base excision repair

AP site formation is a common occurrence in dsDNA. It is the result of spontaneous hydrolysis and the activity of DNA glycosylases as an intermediary step in base excision repair. These AP sites are removed by AP endonucleases, which effect single strand breaks around the site.^[5]

Nucleotide excision repair

V(D)J recombination involves opening stem-loops structures associated with double-strand breaks and subsequently joining both ends. The Artemis-DNAPK_cs complex participates in this reaction. Although Artemis exhibits 5' → 3' ssDNA exonuclease activity when alone, its complexing with DNA-PK_cs allows for endonucleasic processing of the stem-loops. Defects of either protein confers severe immunodeficiency.^[9]

Homologous recombination, on the other hand, involves two

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

See also

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

References