Deoxyribonuclease

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 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. 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. 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 of exonuclease is known as exodeoxyribonucleases. Others cleave anywhere along the chain, known as endodeoxyribonucleases (a subset of endonucleases.) 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.