Deoxyribonuclease (DNase, for short) refers to a group ofglycoproteinendonucleases which areenzymes that catalyze thehydrolytic cleavage ofphosphodiester linkages in theDNA backbone, thus degrading DNA. The role of the DNase enzyme in cells includes breaking downextracellular DNA (ecDNA) excreted byapoptosis,necrosis, andneutrophil extracellular traps (NET) of cells to help reduce inflammatory responses that otherwise are elicited. A wide variety of deoxyribonucleases are known and fall into one of two families (DNase I orDNase II), which differ in theirsubstrate specificities, chemical mechanisms, and biological functions. Laboratory applications of DNase includepurifying proteins when extracted fromprokaryotic organisms. Additionally, DNase has been applied as a treatment for diseases that are caused by ecDNA in the blood plasma. Assays of DNase are emerging in the research field as well.
The two main types of DNase found inanimals are known as deoxyribonuclease I (DNase I) and deoxyribonuclease II (DNase II). These two families have subcategories within them.
The first set of DNases isDNase I. This family consisted of DNase I,DNase1L1,DNase 1L2, andDNase1L3. DNase I cleaves DNA to form twooligonucleotide-end products with 5'-phospho and 3'-hydroxy ends and is produced mainly by organs of thedigestive system. The DNase I family requires Ca2+ and Mg2+cations asactivators and selectively expressed.[1] In terms of pH, the DNAses I family is active in normal pH of around 6.5 to 8.
The second set of DNAases is DNase II. This family consisted of DNase II α and DNase II β. Like DNAase I, DNAase II cleaves DNA to form two oligonucleotide-end products with 5'-hydroxy and 3'-phospho ends. This type of DNAase is more widely expressed in tissues due to high expression in macrophages but limited cell-type expression. Unlike DNAase I, they do not need Ca2+ and Mg2+ cations as activators.[2] In terms ofpH, the DNAase II family is expressed in acidic pH. The cleavage pattern of DNase II is altered in the presence of Dimethyl sulfoxide(DMSO), which significantly affects the structure of DNA.
Although both DNase I and II are glycoprotein endonucleases, DNase I has amonomeric sandwich-type structure with a carbohydrate side chain whereas DNase II has adimericquaternary structure.
DNase I Structure: DNase I is a glycoprotein with a molecular weight of 30,000 Da and a carbohydrate chain of 8-10 residues attached to Asn18 (orange).[3] It is an 𝛼,𝛽-protein with two 6-stranded 𝛽-pleated sheets which form the core of the structure.[4] These two core sheets run parallel, and all others run antiparallel. The 𝛽-pleated sheets lie in the center of the structure while the 𝛼-helices are denoted by the coils on the periphery. DNase I contains four ion-binding pockets, and requires Ca2+ and Mg2+ for hydrolyzing double-stranded DNA.[5] Two of the sites strongly bind Ca2+ while the other two coordinate Mg2+. Little has been published on the number and location of the Mg2+ binding sites, although it has been proposed that Mg2+ is located near the catalytic pocket and contributes to hydrolysis.[6] The two Ca2+ are shown in red in the image. They are bound to DNase I under crystallization conditions and are important for the structural integrity of the molecule by stabilizing the surface loop Asp198 to Thr204 (cyan), and by limiting the region of high thermal mobility in the flexible loop to residues Gly97 to Gly102 (yellow).
DNase II Structure: DNase II contains a homodimeric quaternary structure that is capable of binding double-stranded DNA within a U-shaped clamp architecture. The interior of the U-shaped clamp is largely electropositive, capable of binding negatively-charged DNA. Similar to DNase I, DNase II structure consists of a mixed 𝛼/𝛽 secondary structure with 9 𝛼-helices and 20 𝛽-pleated sheets.[7] Although unlike DNase I, DNase II does not require divalent metal ions for catalysis.[7] The structure consists of protomer A (cyan) and protomer B (green). Each structure consists of two catalytic motifs, which are labeled on protomer B for simplicity: His100 and Lys102 compose the first motif (blue) and His279 and Lys281 compose the second catalytic motif (red).
Some DNases cut, or "cleave", only residues at the ends of DNA molecules. This type ofexonuclease is known asexodeoxyribonucleases. Others cleave anywhere along the chain, known asendodeoxyribonucleases (a subset ofendonucleases.)[8]Some DNases are fairly indiscriminate about theDNA sequence at which they cut, while others, includingrestriction enzymes, are very sequence-specific. Other DNases cleave onlydouble-stranded DNA, others are specific for single-stranded molecules, and others are active toward both.
The action of DNase occurs in three phases. The initial phase introduces multiple nicks in thephosphodiester backbone. The second phase produces acid-soluble nucleotides. The third phase, which is the terminal phase, consists of reduction of oligonucleotides, causing ahyperchromic shift in the UV data.[9]
DNase I predominantly targets double-stranded DNA, and to a lesser extent, some single-stranded DNA for cleavage. DNase I catalyzes nonspecific DNA cleavage by nickingphosphodiester linkages in one of the strands. Its cleavage site lies between the 3′-oxygen atom and the adjacent phosphorus atom, yielding 3′-hydroxyl and 5′-phosphoryloligonucleotides with inversion of configuration at the phosphorus. The DNase enzyme relies on the presence of adivalentcation, which is usually Ca2+, for proper function. The active site of DNase I includes twohistidine residues (His134 and His252) and two acidic residues (Glu78 andAsp 212), all of which are critical for the general acid-base catalysis of phosphodiester bonds.[10]
Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity in the low pH environment of lysosomes where it is typically found in higher eukaryotes. Some forms of recombinant DNase II display a high level of activity in low pH in the absence of divalent metal ions, similar to eukaryotic DNase II.[7] Unlike DNase I, DNase II cleaves the phosphodiester bond between the 5'-oxygen atom and the adjacent phosphorus atom, yielding 3΄-phosphorylated and 5΄-hydroxyl nucleotides.
DNase is commonly used whenpurifying proteins that are extracted fromprokaryotic organisms.Protein extraction often involves the degradation of thecell membrane. It is common for the degraded and fragile cell membrane to belysed, releasing unwanted DNA and the desired proteins. The resulting DNA-protein extract is highly viscous and difficult to purify, in which case DNase is added to break it down.[11] The DNA ishydrolyzed but the proteins are unaffected and the extract can undergo further purification.
Extracellular DNA (ecDNA) is DNA that is found in blood circulation. It appears as a result ofapoptosis,necrosis, orneutrophil extracellular traps (NET)-osis of blood and tissue cells, but can also arise from the active secretion from living cells. EcDNA and their designatedDNA binding proteins are able to activate DNA-sensing receptors,pattern recognition receptors (PRRs). PRRs are able to stimulate pathways that cause an inflammatory immune response. As a result, several studies of inflammatory diseases have found that there are high concentrations of ecDNA in blood plasma. For this reason, DNase has proven to be a possible treatment for the reduction of ecDNA in the blood plasma. DNases can be excreted both intracellularly and extracellularly and can cleave the DNAphosphodiester bond. This function can be used to maintain a low ecDNA concentration, therefore treating inflammation. Illnesses that result from DNA residue in blood have been targeted using the "breaking-down properties" of DNase. Studies have shown DNase to be able to act as a treatment by decreasing the viscosity of mucus.[12][13] Administration of DNase varies dependent on the disease. It can and has been administeredorally, intrapleurally,intravenously,intraperitoneally, and viainhalation.[14] Several studies continue to examine the application of DNase as treatment as well as ways to monitor health. For example, recently, DNase derived frompathogenic bacteria has been used as an indicator for wound infection monitoring.[15]
Cystic fibrosis is agenetic disorder that affects the production of mucus, sweat, and digestive fluids, causing them to become more viscous rather thanlubricant. DNase enzymes can be inhaled using anebulizer bycystic fibrosis sufferers. DNase enzymes help becausewhite blood cells accumulate in the mucus, and, when they break down, they release DNA, which adds to the 'stickiness' of the mucus. DNase enzymes break down the DNA, and the mucus is much easier to clear from the lungs. Specifically, DNase I, also known as FDA approved drugPulmozyme (also known as dornase alfa) is used as a treatment to increase pulmonary function.
Other respiratory illness such asasthma,[16]pleural empyema,[12] andchronic obstructive pulmonary disease have also been found to be positively affected by DNases properties.
Furthermore, recent studies show that intrapleuraltissue plasminogen activator (tPA), a protein that is responsible for the breakdown of blood clots, combined with deoxyribonuclease increase pleural drainage, decreases hospital length of stay, and decreases the need for surgery inparapneumonic effusions andempyema.
Sepsis is a life-threateninginflammatory disease caused by the body's extreme response to an infection. The body begins to attack itself as an inflammatory response encompasses the human body. As a result, high levels of ecDNA have been associated with the bloodstream and therefore, researchers have looked to DNase as an appropriate treatment. Studies have shown that DNase was successful in disrupting NETs and decreasing inflammatory responses. More research on the type and time of administration is needed to further establish DNase as an official treatment.[17][18][19]
Systemic lupus erythematosus (SLE) is anautoimmune disease that results in auto-antibody generation causing inflammation that results in damage to organs, joints, and kidneys. SLE has been linked with low levels of DNase I asapoptotic cells become self-antigens in this disease. DNase I has been investigated as a possible treatment to decrease the amount of apoptotic debris in the human system. It has been suggested that their difficulty might be due to the inability for the enzyme to break down the cell membrane ofchromatin. Studies have shown conflicting results on this treatment, however, further research is being conducted to examine the therapeutic benefits of DNase I.[14][18][20]
Anti-tumor treatment. DNase is known to hold anti-tumor effects due to its ability to break down DNA. High levels of DNA are found to be in cancer patients' blood, suggesting that DNase I might be a possible treatment. There is still a lack of understanding as to why there are such high levels of ecDNA and whether or not DNase will act as an effective treatment. Several mice studies have shown positive results in anti-tumor progression utilizing intravenous DNase I. However, more investigations need to be carried out before being introduced to the public.[21][14]
DNA absorbsultraviolet (UV) light with a wavelength of maximal absorbance near 260 nm. This absorption is due to thepi electrons in thearomatic bases of the DNA. In dsDNA, or even regions ofRNA where double-stranded structure occurs, the bases are stacked parallel to each other, and the overlap of the basemolecular orbitals leads to a decrease inabsorbance of UV light. This phenomenon is called thehypochromic effect. When DNase liberatesnucleotides from dsDNA, the bases are no longer stacked as they are in dsDNA, so that orbital overlap is minimized and UV absorbance increases. This increase in absorbance underlies the basis of the Kunitz unit of DNase activity. One Kunitz unit is defined as the amount of enzyme added to 1 mg/ml salmon sperm DNA that causes an increase in absorbance of 0.001 per minute at the wavelength of 260 nm when acting upon highlypolymerized DNA at 25 °C in a 0.1 M NaOAc (pH 5.0) buffer. The unit's name recognizes the Russian-American biochemistMoses Kunitz, who proposed the standard test in 1946.[22]
A standard enzyme preparation should be run in parallel with an unknown because standardization of DNA preparations and their degree ofpolymerization in solution is not possible.
Single Radial Enzyme Diffusion (SRED)This simple method for DNase I activity measurement was introduced by Nadano et al. and is based on the digestion of DNA in theagarose gel by DNase, which is present in samples punched into the gel.[14] DNase activity is represented by the size of a dispensed circular well in an agarose gel layer, in which DNA stained byethidium bromide is uniformly distributed. After the incubation, a circular dark zone is formed as the enzyme diffuses from the well radially into the gel and cleaves DNA. SRED underwent many modifications, which led to an increase in sensitivity and safety, such as the replacement of ethidium bromide withSYBR Green I or other DNA gel stains.[23]
Colorimetric DNase I Activity Assay
Kinetic colorimetric DNase I activity assay is developed for the assessment of the stability of the human recombinant DNase I (Pulmozyme). The method was adjusted from a colorimetric endpoint enzyme activity assay based on the degradation of a DNA/methyl green complex.[24]
Extracellular deoxribonucleases act asvirulence factors for pathogens, includingPlasmodium[25] and severalStreptococcus species,[26][27] as they allow the pathogens to defend themselves againstneutrophil extracellular traps which consist primarily of histone-linkedextracellular DNA.[28][29]
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