Nuclease
Nucleases are ubiquitous phosphodiesterase enzymes that cleave phosphodiester bonds within nucleic acid molecules. They are indispensable for the both cellular and viral development. Nucleases that are specific for DNA are called deoxyribonucleases (DNases), while nucleases that recognize RNA (RNases) are called ribonucleases. There are many distinct types, all of which function to recognize a wide range of different yet types of molecules. Some nucleases, however, cleave nonspecific nucleic acid substrates. They also provide a variety of protective mechanisms. For example, they are responsible for degrading host cell DNA following viral infection and they are essential to DNA repair mechanisms such as nucleotide excision repair. Other important functions within the cell that nucleases play a role include DNA synthesis, DNA recombination, maturation of RNAs or RNA splicing, and DNA packaging involving either chromosomes or viral compartments. While some nucleases serve to degrade exposed single-stranded DNA, others are specific for double-standed DNA.
Exonucleases can remove nucleotides starting from the free ends of DNA inward, while endonucleases target somewhere within a DNA. Exonucleases can also operate unidirectionally in either the 5' (five prime) to 3' (three prime) direction or vice versa.
Targeting of these enzymes to the appropriate site can be accomplished either with the help of specific proteins or by recognizing specific sequences. Endonucleases can affected by how the chromosome is packaged. For example, there are regions along the chromosome that are less tightly coiled around nucleosomes, making them vunerable to nuclease attack. These localized areas along the eukaryotic chromosomes are called nuclease hypersensitivity sites.
The field of molecular biology has greatly benefited from the identification and characterization of nucleases. Although many biologists aim to preserve DNA sequences, DNAases can be used in protocols that benefit from the removal or alteration of DNA. For example, DNases can be used for eliminating DNA during RNA isolations, identifying DNA-protein interactions (called DNase footprinting), or by creating nicks in the DNA prior to radiolabeling by nick translation. Rnase A is the most common endonuclease that degrades RNA and can be used to reduce RNA contamination in DNA isolation or plasmid preparations. It can also be used in mapping mutations in RNA by cleaving RNA in RNA:DNA hybrids where there are single base mismatches. The most common type of nucleases used in the laboratory are restriction enzymes. Restriction enzymes are nucleases that recognize specific DNA sequences and provide a powerful tool in recombinant DNA technology. They can be used to digest DNA and clone the fragments for sequencing purposes. Additionally, they are used to identify differences in the sequences of two alleles that create restriction fragment length polymorphisms (RFLPs) when digested.
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