“DNase is the group of DNA enzymes and a type of nuclease used for hydrolytic cleaving of the DNA. It has lucrative applications in biological science and therefore has great experimental values.
DNase short for Deoxyribonuclease is the class of enzymes having unmatched importance in both in vivo and in vitro experiments. Cleaving DNA either, on ends or in between is the function it performs.
Consequently, it releases tension during DNA replication in vivo. DNase cleaves the DNA for various gene manipulation and gene editing experiments. It is a class of “nuclease” either endonuclease or exonuclease and is broadly classified into two major groups; DNase I and DNase II.
It is believed that the DNase is evolved initially in eukaryotes to destroy foreign bacteria (pathogenic DNA), it’s part of the defense, although no hypothesis yet clearly defined how DNase is evolved.
Though the role of DNase has been well studied for in vivo processes how it works for genetic experiments and gene therapy processes is yet not understood by some. So in this article, I will explain the importance of DNase and how it is used in certain experiments. We will also look into applications as well.
The goal of this article is to educate students and provide affirmative knowledge to research students. I hope I will add value.
What is DNase?
DNase is a class of nuclease that cleaves or cut DNA at precise or random locations. It’s a type of protein, performs the catalytic reaction. The encoding gene for DNase is DNASE.
For different DNases, separate DNASE genes are located on different chromosomes. Each one is different in terms of length and number of exons as well. As we illustrated, there are two broad classes of DNase; DNase I and DNase II. We will discuss the function of each one in the upcoming section.
“DNase is a nuclease that hydrolyzes either extracellular or intracellular DNA by catalytic reaction.”
Mechanism of action:
A type of nucleic acid DNA, deoxyribonucleotides are either dsDNA or ssDNA that can be cleaved by DNase through a catalytic reaction. Structurally, two adjacent nucleotides are joined by the phosphodiester linkage and form a chain of nucleotides, known to us as a polynucleotide chain.
DNase hydrolyzes the phosphodiester bond and breaks or cleaves the DNA either on ends or in-between. Cleaving produces 3’- hydroxyl and 5’ phosphate ends or vice versa, which depends on the type of DNase.
Ends generated by cleaving are either sticky ends or blunt ends. The intracellular DNase cleaves intracellular DNA whilst the extracellular DNase cleaves free DNA fragments.
Classification of DNase:
Ideally, DNases are classified into two broad categories; DNase I and DNase II each one is classified into other subcategorize given below,
|Type of DNase
|DNase I, DNase1L3, DNase1L2, DNase1L1
|DNase IIα, DNaseIIβ, L-DNase II
DNase I cleave the normal B-form DNA more effectively than other forms hence is widely present and used in common genetic experiments.
The enzyme is encoded by a gene DNASE I which is located on chromosome 16 at 16p13.3. It has 17 fully functional exons encode the DNase I. The molecular weight of the DNase I protein is 38KDa having optimum pH of 6.5 to 8.
It requires co-factors such as bivalent Mg2+ and Ca2+, note that all other enzymes of the present family also need cofactors to enhance their activity. The pancreas secretes DNase I.
The two major functions Mg2+ and Ca2+ perform are catalytic cleavage and maintenance of the enzyme, respectively.
Importantly, EDTA, G-actin and EGTA are three chelating agents that inhibit the activity of the present enzyme. DNase I sub-classified into DNase1L3, DNase1L2, DNase1L1.
On cleaving, it produces 5’-phosphate and 3’-hydroxy ends. The dsDNA (Double-stranded DNA) is the main substrate for the present form of DNase as it cleaves the dsDNA 500 fold more accurately and effectively than ssDNA.
|Location on chromosome
The physiological role of DNase I is to reduce autoimmune reactions by digesting major extracellular nucleoproteins. Systemic lupus erythematosus, a kind of autoimmune disease is caused by a mutation in the DNASE1 gene, which reduces the activity of the DNase enzyme, gradually.
Often known as “acid DNase” the DNase II group of nucleases have optimum pH of 4.8 to 5.2. The catalytic activity of the present enzyme is too low in comparison to the DNase I and also decreases as the pH increases.
Moreover, co-factors such as Mg2+ or Ca2+ aren’t required by it. Instead, the presence of salts such as calcium, zinc or magnesium strongly decreases the catalytic activity.
The molecular weight of the Dnase II is 43KDa and is produced by the spleen, majorly. In addition to this, G-actin and chelating agents such as EDTA or EGTA can’t inhibit the activity of DNase II.
On hydrolysis, it produces 3’-Phospho and 5’-hydroxy ends.
|DNase II alpha
|DNase II beta
Here a specialized DNase II alpha has a specific function to cleave both single-strands DNA of dsDNA and hence also known as nicking endonuclease or nicking DNase.
This endonuclease is present intracellularly in all human cells but also present in fluids like saliva, testicular liquid, and blood in small amounts. Noteworthy, the enzyme DNase II alpha never participates in DNA metabolisms such as replication, recombination and repair.
The alpha form is also known as the lysosome enzyme as it is mainly present in lysosomes. The type of DNase II alpha and DNase II beta occurs by the mechanism of gene duplication of the DNASE II gene.
Importantly, the acid DNase- DNase II beta is only present in the lens of eyes protects from cataracts by cleaving DNAs.
Degradation of bacterial DNA and activation of Toll-like receptor 9.
The pivotal function of this enzyme is to cleave or degrade bacterial DNA to protect the host and activating of Toll-like receptor 9.
However, the function of L-DNase II is totally different. Here the L-DNase II degrade DNA during the process of apoptosis. This means it induces cell death, apoptosis is the process of cell death.
Measuring DNase activity:
The spectrophotometric method described by Kunitz was the first method to measure the activity of DNase in 1950. To honor the original researcher, the unit to measure the activity of DNase is named “Kunitz”.
When the enzyme is added to the solution of dsDNA, the absorbance of UV light increases at 260nm wavelength.
DNA absorbs UV light at 160nm, the dsDNA absorbs less UV light as bases are tightly stacked. When the DNase is added to the solution of dsDNA, it starts degrading followed by increments in the UV absorbance.
Depending upon the absorbance spectra, the activity can be measured.
The Kunitz unit is defined as,
“The amount of enzyme added to 1mg/m salmon sperm DNA.”
Another method to detect the activity of DNase is known as SRED- Single Radial Enzyme Diffusion, which is more effective, accurate and reliable.
The present method is based on agarose gel electrophoresis, here when DNase is applied to the gel, it degrades DNA (present in a gel) by catalytic reaction. As it cleaves more DNA, a circular zone of activity is observed under the UV light when EtBr stains the gel.
The circle around the DNA sample indicates enzymatic activity which can be readily measured, the activity of enzyme determined by this technique as well.
Fluorometric and colorimetric assay, ELISA are other techniques, also readily available nowadays.
Applications of DNase:
DNase cleaves DNA; either dsDNA or ssDNA, and consequently, is used in a variety of assays and applications as a scissor. In addition, Its role as a treatment is significant.
The present enzyme is used as a treatment agent in cystic fibrosis and other diseases. It is used by inhaling the sample of DNase to treat the disease.
FDA- food and drug administration approved the inhalation therapy to treat respiratory disorders by the use of DNase. Here when the DNase- an artificial, genetically synthesized one is given through inhalation, it causes a positive effect in the case of cystic fibrosis.
This is the reason it is widely used in pulmonary diseases.
Another important application of it is genetic engineering technology. Here it is used as a scissor to cleave DNA for various experiments such as gene editing, gene manipulation, gene therapy, gene transfer and CRISPR-CAS9.
The CAS9 is also a type of nuclease that helps to cleave the DNA at specific locations.
One of the most fascinating applications of DNase is in RNAi studies and RNA extraction. Here to purify the RNA sample, the contaminant DNA is degraded and removed using the DNase.
DNase has significant importance in genetic engineering as nearly all experiments of gene manipulations are not possible without it. Commercially available DNase is powerful enough to cleave the target even at the right location, precisely and effectively.
Keep in mind to not use DNase during DNA isolation, it immediately cleaves DNA and reduces the yield.
Also, do not store DNase with other important DNA samples or PCR products.
Suck D. (1994). DNA recognition by DNase I. Journal of molecular recognition: JMR, 7(2), 65–70. https://doi.org/10.1002/jmr.300070203.