Nuclease is the term used for an enzyme that cleaves nucleic acids. They are also known as nucleodepolymerase or polynucleotide. Nucleases belong to the enzyme class hydrolases and perform a specific role. The ribonucleases work on the ribonucleic acids (RNA) and deoxyribonucleases work on the deoxyribonucleic acids (DNA).
Some enzymes that have a general role to play such as phosphodiesterases, which hydrolyze phosphoric acid esters, can also be termed nucleases because nucleic acids are affected by their action. Nucleases are present in plants as well as animals.
Nuclease has the role of cleaving the phosphodiester bonds between nucleotides of nucleic acids and causing single and double-stranded breaks in their target molecules. They are essential in living organisms for their several DNA repair aspects. If there are defects in certain nucleases, it can cause immunodeficiency or genetic instability. Nucleases also play a critical role in molecular cloning.
Nuclease can be classified into two broad categories based on the locus of activity: Exonucleases and Endonucleases. While Exonucleases digest nucleic acids from the ends, the Endonucleases work on regions in the centre of target molecules. These categories can be further subcategorized into deoxyribonucleases and ribonucleases. The deoxyribonucleases (DNA nuclease) act on DNA, while the ribonucleases work on RNA.
Micrococcal nuclease is a type of endonuclease that primarily digests single-stranded DNA or RNA, specifically at AU- or AT-rich regions. This enzyme also digests double-stranded DNA or RNA and is a critical component of chromatin immunoprecipitation (ChIP) assays. Micrococcal Nuclease can digest 5'-phosphodiester bonds of RNA and DNA, and yields 3'-phosphate mononucleotides and oligonucleotides. It needs Ca2+ as a cofactor for performing its function and gets completely inactivated by EGTA or EDTA.
Mung Bean Nuclease
Mung bean nuclease is a single-stranded (ssDNA or RNA) endonuclease that helps in the removal of single-stranded extension in double-stranded DNA. It can be used for removing both 3' and 5' single-stranded overhangs from double-stranded DNA for the creation of blunt ends. It cleaves single-stranded RNA and DNA, cleaves the single-stranded region in a DNA hairpin and helps in the mapping of RNA transcripts.
Before it can cleave the molecule, a nuclease must associate with a nucleic acid. It requires a certain degree of recognition and nucleases use both specific and nonspecific associations in their means of binding and recognition. Both modes or means play a critical role in living organisms, especially in the area of DNA repair.
Nonspecific endonucleases that are involved in DNA can scan DNA for damage or target sequence. This type of nuclease diffuses along with the DNA until it meets a target. Then, the residues of its active site mingle with the DNA chemical groups. For endonucleases such as BamHI, EcoRV, and PvuII, electrostatic interactions between the DNA and minimal surface area of the protein are involved in the nonspecific binding. The overall shape of the DNA is undeformed because of this weak association, thus remaining in B-form.
Site-specific nucleases can form associations that are much stronger in contrast. They can draw DNA into the deep groove of their DNA-binding domain. It causes a significant deformation of the DNA tertiary structure and is accomplished with basic residue-rich or positively charged surfaces. Such nucleases engage in electrostatic interaction with the DNA extensively.
Some nucleases involved in DNA repair are partial sequence-specific. The majority of the nucleases however are nonspecific and recognize structural abnormalities generated in the DNA backbone with the use of base pair mismatches.
1. What are Restriction Nucleases?
Ans. Restriction nucleases are enzymes that split the DNA molecules in which they can recognize particular subunits. While the target DNA molecule is split at random sites by some nucleases (Type I), the others split the DNA molecule only at the recognition site (Type II). In some cases, the DNA molecules are split at a fixed distance from the recognition site (Type III). Type II and III restriction enzymes are used extensively in the elucidation of the bases sequence in DNA molecules. They are also used in recombinant genetic engineering or DNA technology. There are more than sequence-specific and non-specific restriction enzymes. They have been isolated from more than 230 strains of bacteria since HindII was discovered.
The names of these restriction enzymes reflect their origin: the first letter of the name is derived from the genus and the second two letters from the prokaryotic cell species from which they were isolated. For example, EcoRI has been derived from Escherichia coli RY13 bacteria, HindII comes from Haemophilus influenzae strain Rd. The numbers after the nuclease names denote the order of isolation of the enzymes from the single strains of bacteria: EcoRI, EcoRII.
2. What is Benzonase?
Ans. Benzonase is a genetically produced endonuclease that attacks all forms of DNA and RNA (single-stranded, double-stranded, circular, and linear) and degrades them. It is ideal for removing nucleic acids from recombinant proteins. It is effective over a large range of operating scenarios and for applications that require complete digestion of nucleic acids. Benzonase decreases viscosity in protein extracts and restricts cell clumping.
3. What are Meganucleases?
Ans. The length of the nuclease's recognition sequence and the complexity of the DNA determine the frequency at which a particular nuclease will cut a given DNA molecule. A longer recognition sequence results in less frequent digestion because of the statistical chance of finding the bases in a particular order randomly. For instance, the occurrence prediction for a given four-base sequence (that corresponds to the recognition site for a hypothetical nuclease) is going to be every 256 base pairs on average (where 44 = 256), but for any given six-base sequence it would be once every 4,096 base pairs on average (46 = 4096)
Meganuclease is a unique family of nucleases characterized by having larger, and less common, recognition sequences that consist of 12 to 40 base pairs. These nucleases are especially useful for Genome engineering and genetic engineering applications in complex organisms such as mammals and plants. These are cases where typically larger genomes (billions of base pairs) would result in frequent site-specific digestion with the help of traditional nucleases.