What Is DNase?
Deoxyribonuclease (DNase) is a class of nuclease enzymes that cleaves the DNA molecule. It breaks the phosphodiester bond formed between two nucleotides of dsDNA or ssDNA, by cleaving either at/on ends or between the nucleic acid strands.
The enzyme is encoded by two different DNase genes—different in length and number of exons—and thus, is of two types: DNase I and DNase II.
Based on the type of DNase that acts on the genomic DNA, they either produce 3’ hydroxyl and 5’ phosphate ends or vice versa, with either a sticky or blunt end, after the cleaving process.
Moreover, based on the presence of DNase in a particular location, it’s classified into two groups:
- Extracellular DNase: Found outside of organisms in any type of environmental sample, abundant in a variety of habitats including soil, oceans, sediments, and freshwater.
- Intracellular DNase: Found within the cell membranes of organisms.
DNase enzymes prefer different parameters to cleave nucleic acid sequences:
- Some cut anywhere within the strand and some cleave at the end.
- Some DNase cleaves any sequence while some cut at a specific sequence.
- Some DNases cleave double-stranded DNA while others single-stranded DNA.
While DNase has many essential roles in performing cellular functions, their activities are modified and altered by using DNase inhibitors.
DNase is also one of the essential reagents in labs to perform a spectrum of lucrative applications in molecular biology and biological science to obtain great experimental values.
In this article, we will cover more about the types of DNase enzymes, their applications, and how the activity of DNases can be measured in labs.
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Types of DNase
DNase is of two types based on the type of DNA substrate, their working mechanisms, and the resulting product at the end of cleavage.
This type of DNase is an endonuclease encoded by the gene DNASE1 and secreted by the pancreas. It acts on ssDNA, dsDNA, and chromatin. It non-specifically cuts these molecules adjacent to pyrimidine nucleotides, which results in the formation of 5′ phosphate-terminated polynucleotides with a free 3’ hydroxyl group.
The enzyme is sub-classified into DNase1L3, DNase1L2, and DNase1L1. It requires bivalent cofactors such as Mg2+ and Ca2+ to perform the catalytic functions and enhance their enzymatic activity. Some inhibitors of the enzyme that hinder its activity include G-actin, EDTA, and EGTA.
The functional role of DNase I is to digest extracellular nucleoproteins resulting in reduced autoimmune reactions and DNA fragmentation during apoptosis. Any mutation in the enzyme results in the gradual reduction of DNase enzymatic activity.
In labs, the enzyme is used for amplification of cDNA, DNA manipulation, and creating DNA fragments or oligonucleotides for in vitro recombination studies.
This type of DNase is also known as acid DNase and has optimum pH of 4.8 to 5.2, above or below which its enzymatic activity is reduced. The catalytic functions of the enzyme are lower in comparison to DNase I. It can cleave both the strands of dsDNA and is thus also referred to as nicking DNase.
This type of DNase does not require bivalent cofactors, such as Mg2+ or Ca2+, to perform its functions, but the presence of salts like zinc, calcium, and magnesium decreases its activity. Moreover, their functions are also inhibited by EDTA, EGTA, and G-actin like DNase I.
There are two types of DNase II, alpha and beta. In addition to its presence in all human cells, the enzyme is also present in fluids, such as testicular liquid, saliva, and blood in small amounts.
DNase II alpha is ubiquitously expressed in most human tissues and contained in lysosomes, playing an important role in the metabolism and protection of organisms. DNase II beta is mainly expressed in the salivary glands and in the eye where it protects the lenses from cataracts by fragmenting DNA.
Applications of DNase
DNase is used in several lab assays as scissors, especially for the applications of developing treatments for severe diseases. For example, it’s used as a treatment for cystic fibrosis and some respiratory disorders.
Based on the goal of experiments, DNase is involved in many assays like in vitro transcription, real-time PCR, mRNA decay assay, and reverse transcription.
- Preparation of DNA-free RNA
- Removal of template DNA after in vitro transcription
- DNA labeling by nick-translation
- RNase-free footprinting
- Preparation of DNA-free RNA before RT-qPCR and RT-PCR
- RNA analysis and In vitro Synthesis (IVT)
- microRNA profiling
- Labeling antibodies
- Plasmid preparation and purification
- Molecular cloning and DNA sequencing
- Understanding mitochondrial promoter recognition in humans and other mammals
The commercially available RNase-free DNase enzymes are generally recombinant enzymes that are artificially created by researchers for lab applications. For example, some companies offer products manufactured from E.coli having an MBP fusion clone of Bovine Pancreatic DNase I.
They are designed to serve specific purposes, information about which can be found on the safety data sheet (SDS), certificate of analysis, and materials safety data sheet that comes with the product.
The enzyme requires proper conditions to perform its functions in labs, such as:
- 1X DNase I Reaction Buffer (prepared by using reagents like 10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, (pH 7.6 at 25°C). Any disturbance in the buffer condition, such as an increase in NaCl or KCL salt concentration disturbs the enzymatic activity of enzymes.
- Incubation at 37°C
Measuring DNase I Activity with Assays
The wavelength at which DNA absorbs UV light is near 260 nm. In dsDNA, at the regions of double-stranded structures, bases are stacked parallel to each other, which causes overlapping of molecular orbitals and a decrease in absorbance of UV light. It is known as the hypochromic effect.
Similarly, when the double-stranded structure is distorted, the bases are no longer stacked and the orbital overlap is reduced, which leads to an increase in the UV absorbance and forms the basis of the Kunitz unit of DNAse activity.
One Kunitz unit is defined as the amount of enzyme required to completely degrade 1 µg of plasmid DNA in 10 minutes at 37℃.
This technique is widely used in labs for high-throughput screening, mainly because of the involvement of a diverse selection of fluorophores, ease of operation, high sensitivity, and various readout modes.
However, the success of the technique in measuring the enzymatic activity depends on several factors including:
- the careful choice of excitation (Ex) and emission (Em) wavelengths
- the selection of flexible and sensitive optics
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DNases belong to a class of enzymes that are involved in the cleavage of DNA molecules. They do so by breaking the phosphodiester bond between the two nucleotides.
Based on the substrate they use, the working mechanism, and the resulting product at the end of their catalytic reactions, DNases are classified into two groups: DNase I and DNase II.
Apart from their metabolic roles in organisms, such as apoptosis and protecting organisms against diseases, these enzymes have major roles in laboratory applications.
In labs, they are used in assays such as cloning, sequencing, and different PCR reactions. However, the success of these molecular biology experiments demands the need for ultra-high purity reagents free from nucleases and proteases contaminants.
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