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Class of enzymes which cleave nucleic acids

In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect 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).

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.

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.]]

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. Nonspecific endonucleases involved in DNA repair can scan DNA for target sequences or damage. Such a nuclease diffuses along DNA until it encounters a target, upon which the residues of its active site interact with the chemical groups of the DNA. In the case of endonucleases such as EcoRV, BamHI, and PvuII, this nonspecific binding involves electrostatic interactions between minimal surface area of the protein and the DNA. This weak association leaves the overall shape of the DNA undeformed, remaining in B form.

Many forms of DNA damage stop progression of the replication fork, causing the DNA polymerases and associated machinery to abandon the fork. It must then be processed by fork specific proteins. The most notable is MUS81. Deletions of which causes UV or methylation damage sensitivity in yeast, in addition to meiotic defects.

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 MutS, MutL, and MutH) corrects point mutations and small turns. MutS recognizes and binds to mismatches, where it recruits MutL and MutH. MutL mediates the interaction between MutS and MutH, and enhances the endonucleasic activity of the latter. MutH recognizes hemimethylated 5'—GATC—3' sites and cleaves next to the G of the non methylated strand (the more recently synthesized strand). VSP repair is initiated by the endonuclease Vsr. It corrects a specific T/G mismatch caused by the spontaneous deamination of methylated cytosines to thymines. Vsr recognizes the sequence 5'—CTWGG—3',
where it nicks the DNA strand on the 5' side of the mismatched thymine (underlined in the previous sequence). One of the exonucleases RecJ, ExoVII, or ExoI then degrades the site before DNA polymerase resynthesizes the gap in the strand. V(D)J recombination involves opening stem loops structures associated with double strand breaks and subsequently joining both ends. The Artemis DNAPKcs complex participates in this reaction. Although Artemis exhibits 5' → 3' ssDNA exonuclease activity when alone, its complexing with DNA PKcs allows for endonucleasic processing of the stem loops. Defects of either protein confers severe immunodeficiency. Homologous recombination, on the other hand, involves two homologous DNA duplexes connected by D loops or Holliday junctions. In bacteria, endonucleases like RuvC resolve Holliday junctions into two separate dsDNAs by cleaving the junctions at two symmetrical sites near the junction centre. In eukaryotes, FEN1, XPF ERCC1, and MUS81 cleave the D loops, and Cce1/Ydc2 processes Holliday junctions in mitochondria.

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.