Дезоксирибонуклеазы: структура, функции и применение.

Enzymes that break down DNA
Deoxyribonuclease (DNase, for short) refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells includes breaking down extracellular DNA (ecDNA) excreted by apoptosis, necrosis, and neutrophil 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 or DNase II), which differ in their substrate specificities, chemical mechanisms, and biological functions. Laboratory applications of DNase include purifying proteins when extracted from prokaryotic 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 in animals are known as deoxyribonuclease I (DNase I) and deoxyribonuclease II (DNase II). These two families have subcategories within them.

The first set of DNases is DNase I. This family consisted of DNase I, DNase1L1, DNase 1L2, and DNase1L3. DNase I cleaves DNA to form two oligonucleotide end products with 5’ phospho and 3’ hydroxy ends and is produced mainly by organs of the digestive system. The DNase I family requires Ca2+ and Mg2+ cations as activators and selectively expressed. 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 ɑ and DNase II ꞵ. 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. In terms of pH, 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 a monomeric sandwich type structure with a carbohydrate side chain whereas DNase II has a dimeric quaternary 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). It is an 𝛼,𝛽 protein with two 6 stranded 𝛽 pleated sheets which form the core of the structure. 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. 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. 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. Although unlike DNase I, DNase II does not require divalent metal ions for catalysis. Some DNases are fairly indiscriminate about the DNA sequence at which they cut, while others, including restriction enzymes, are very sequence specific. Other DNases cleave only double 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 the phosphodiester backbone. The second phase produces acid soluble nucleotides. The third phase, which is the terminal phase, consists of reduction of oligonucleotides, causing a hyperchromic shift in the UV data.

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 nicking phosphodiester 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′ phosphoryl oligonucleotides with inversion of configuration at the phosphorus. The DNase enzyme relies on the presence of a divalent cation, which is usually Ca2+, for proper function. The active site of DNase I includes two histidine residues (His134 and His252) and two acidic residues (Glu78 and Asp 212), all of which are critical for the general acid base catalysis of phosphodiester bonds.

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. The DNA is hydrolyzed 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 of apoptosis, necrosis, or neutrophil extracellular traps (NET) osis of blood and tissue cells, but can also arise from the active secretion from living cells. EcDNA and their designated DNA 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 DNA phosphodiester 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. Administration of DNase varies dependent on the disease. It can and has been administered orally, intrapleurally, intravenously, intraperitoneally, and via inhalation. Several studies continue to examine the application of DNase as treatment as well as ways to monitor health. For example, recently, DNase derived from pathogenic bacteria has been used as an indicator for wound infection monitoring.