[Biology Class Notes] on Nuclease Pdf for Exam

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.

Function of Nucleases

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.

Types of Nucleases

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

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.

Site Recognition

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.