Deoxyribonuclease (DNase, for short) refers to a group of
glycoprotein
Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycos ...
endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
s which are
enzymes
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
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
Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
,
necrosis
Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated dige ...
, and
neutrophil extracellular traps
Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens. Neutrophils are the immune system's first line of defense against infection and have conventionally bee ...
(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
Deoxyribonuclease I (usually called DNase I), is an endonuclease of the DNase family coded by the human gene DNASE1.
DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding ...
or
DNase II
Deoxyribonuclease II (, ''DNase II'', ''pancreatic DNase II'', ''deoxyribonucleate 3'-nucleotidohydrolase'', ''pancreatic DNase II'', ''acid deoxyribonuclease'', ''acid DNase'') is an endonuclease that hydrolyzes phosphodiester linkages of deoxyr ...
), which differ in their
substrate specificities, chemical mechanisms, and biological functions. Laboratory applications of DNase include
purifying proteins when extracted from
prokaryotic
A prokaryote () is a Unicellular organism, single-celled organism that lacks a cell nucleus, nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek language, Greek wikt:πρό#Ancient Greek, πρό (, 'before') a ...
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.
Types
The two main types of DNase found in
metazoans
Animals are multicellular, eukaryotic organisms in the biological kingdom Animalia. With few exceptions, animals consume organic material, breathe oxygen, are able to move, can reproduce sexually, and go through an ontogenetic stage in ...
are known as deoxyribonuclease I (DNase I) and deoxyribonuclease II (DNase II). These two families have subcategories within them.
The DNase I Family: DNase I, DNase1L1, DNase 1L2, DNase1L3
The first set of DNases is
DNase I
Deoxyribonuclease I (usually called DNase I), is an endonuclease of the DNase family coded by the human gene DNASE1.
DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding ...
. This family consisted of DNase I,
DNase1L1
Deoxyribonuclease-1-like 1 is an enzyme that in humans is encoded by the ''DNASE1L1'' gene.
This gene encodes a member of the deoxyribonuclease family and the protein shows high sequence similarity to lysosomal
A lysosome () is a membrane-bou ...
,
DNase 1L2, and
DNase1L3
Deoxyribonuclease gamma is an enzyme that in humans is encoded by the ''DNASE1L3'' gene.
This gene encodes a member of the DNase family. The protein hydrolyzes DNA, is not inhibited by actin, and mediates the breakdown of DNA during apoptosis. Alt ...
. DNase I cleaves DNA to form two
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...
-end products with 5’-phospho and 3’-hydroxy ends and is produced mainly by organs of the
digestive system
The human digestive system consists of the gastrointestinal tract plus the accessory organs of digestion (the tongue, salivary glands, pancreas, liver, and gallbladder). Digestion involves the breakdown of food into smaller and smaller compone ...
. The DNase I family requires Ca2+ and Mg2+
cations
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
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 DNase II Family: DNase II ɑ and DNase II ꞵ
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
Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula ( CH3)2. This colorless liquid is the sulfoxide most widely used commercially. It is an important polar aprotic solvent that dissolves both polar and nonpolar compounds an ...
), which significantly affects the structure of DNA.
Structure
Although both DNase I and II are glycoprotein endonucleases, DNase I has a
monomer
In chemistry, a monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization.
Classification
Mo ...
ic sandwich-type structure with a carbohydrate side chain whereas DNase II has a
dimeric quaternary structure
Protein quaternary structure is the fourth (and highest) classification level of protein structure. Protein quaternary structure refers to the structure of proteins which are themselves composed of two or more smaller protein chains (also refe ...
.
![DNase 1 image 1](https://upload.wikimedia.org/wikipedia/commons/5/53/DNase_1_image_1.png)
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 Ca
2+ and Mg
2+ for hydrolyzing double-stranded DNA.
Two of the sites strongly bind Ca
2+ while the other two coordinate Mg
2+. Little has been published on the number and location of the Mg
2+ binding sites, although it has been proposed that Mg
2+ is located near the catalytic pocket and contributes to hydrolysis. The two Ca
2+ 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](https://upload.wikimedia.org/wikipedia/commons/5/58/DNase_II.png)
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).
Mechanism
Some DNases cut, or "cleave", only residues at the ends of DNA molecules. This type of
exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the ...
is known as
exodeoxyribonuclease Exodeoxyribonucleases are both exonucleases and deoxyribonucleases. They catalyze digestion of the ends of linear DNA. They are a type of esterase. They are classified EC 3.1.11.
See also
* Deoxyribonuclease Deoxyribonuclease (DNase, for short) r ...
s. Others cleave anywhere along the chain, known as
endodeoxyribonuclease Endodeoxyribonuclease are both endonucleases and deoxyribonucleases. They catalyze cleavage of the phosphodiester bonds in DNA. They are classified with EC numbers 3.1.21 through 3.1.25.
Examples include:
* DNA restriction enzymes
* micrococcal ...
s (a subset of
endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
s.)
Some DNases are fairly indiscriminate about the
DNA sequence
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
at which they cut, while others, including
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
s, 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 Mechanism
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
In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...
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
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...
s with inversion of configuration at the phosphorus. The DNase enzyme relies on the presence of a
divalent
In chemistry, the valence (US spelling) or valency (British spelling) of an element is the measure of its combining capacity with other atoms when it forms chemical compounds or molecules.
Description
The combining capacity, or affinity of an ...
cation
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
, which is usually Ca
2+, for proper function. The active site of DNase I includes two
histidine
Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the de ...
residues (His134 and His252) and two acidic residues (
Glu78 and
Asp
Asp may refer to:
Places
* Asp, part of Densbüren, Aargau, Switzerland
* Aspe (''Asp'' in Valencian), Alicante, Spain
* Asp Lake, a lake in Minnesota
Animals
* Asp (fish)
* Asp (snake), in antiquity, one of several venomous snakes
** ''Cera ...
212), all of which are critical for the general acid-base catalysis of phosphodiester bonds.
DNase II Mechanism
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.
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.
Applications
Laboratory applications
DNase is commonly used when
purifying proteins that are extracted from
prokaryotic
A prokaryote () is a Unicellular organism, single-celled organism that lacks a cell nucleus, nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek language, Greek wikt:πρό#Ancient Greek, πρό (, 'before') a ...
organisms.
Protein extraction often involves the degradation of the
cell membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
. It is common for the degraded and fragile cell membrane to be
lysed, 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. The DNA is
hydrolyzed
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile.
Biological hydrolysis ...
but the proteins are unaffected and the extract can undergo further purification.
Treatment
Extracellular DNA (ecDNA) is DNA that is found in blood circulation. It appears as a result of
apoptosis
Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
,
necrosis
Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated dige ...
, or
neutrophil extracellular traps
Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens. Neutrophils are the immune system's first line of defense against infection and have conventionally bee ...
(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 receptor
Pattern recognition receptors (PRRs) play a crucial role in the proper function of the innate immune system. PRRs are germline-encoded host sensors, which detect molecules typical for the pathogens. They are proteins expressed, mainly, by cells of ...
s (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
In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is ...
. 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
Intravenous therapy (abbreviated as IV therapy) is a medical technique that administers fluids, medications and nutrients directly into a person's vein. The intravenous route of administration is commonly used for rehydration or to provide nutrie ...
,
intraperitoneally
Intraperitoneal injection or IP injection is the injection of a substance into the peritoneum (body cavity). It is more often applied to animals than to humans. In general, it is preferred when large amounts of blood replacement fluids are needed ...
, and via
inhalation
Inhalation (or Inspiration) happens when air or other gases enter the lungs.
Inhalation of air
Inhalation of air, as part of the cycle of breathing, is a vital process for all human life. The process is autonomic (though there are exceptions ...
.
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
Pathogenic bacteria are bacteria that can cause disease. This article focuses on the bacteria that are pathogenic to humans. Most species of bacteria are harmless and are often Probiotic, beneficial but others can cause infectious diseases. The n ...
has been used as an indicator for wound infection monitoring.
Respiratory diseases
Cystic fibrosis
Cystic fibrosis (CF) is a rare genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. Long-term issues include difficulty breathing and coughing up mucus as a result of frequent lung infections. O ...
is a
genetic disorder
A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders ...
that affects the production of mucus, sweat, and digestive fluids, causing them to become more viscous rather than
lubricant
A lubricant (sometimes shortened to lube) is a substance that helps to reduce friction between surfaces in mutual contact, which ultimately reduces the heat generated when the surfaces move. It may also have the function of transmitting forces, t ...
. DNase enzymes can be inhaled using a
nebulizer
In medicine, a nebulizer (American English) or nebuliser (British English) is a drug delivery device used to administer medication in the form of a mist inhaled into the lungs. Nebulizers are commonly used for the treatment of asthma, cystic fibro ...
by
cystic fibrosis
Cystic fibrosis (CF) is a rare genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. Long-term issues include difficulty breathing and coughing up mucus as a result of frequent lung infections. O ...
sufferers. DNase enzymes help because
white blood cell
White blood cells, also called leukocytes or leucocytes, are the cell (biology), cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and de ...
s 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 drug
Pulmozyme
Dornase alfa (proprietary name Pulmozyme from Genentech) is a highly purified solution of recombinant human deoxyribonuclease I (rhDNase), an enzyme which selectively cleaves DNA. Dornase alfa hydrolyzes the DNA present in sputum/mucus of cy ...
(also known as dornase alfa) is used as a treatment to increase pulmonary function.
Other respiratory illness such as
asthma
Asthma is a long-term inflammatory disease of the airways of the lungs. It is characterized by variable and recurring symptoms, reversible airflow obstruction, and easily triggered bronchospasms. Symptoms include episodes of wheezing, cou ...
,
pleural empyema
Pleural empyema is a collection of pus in the pleural cavity caused by microorganisms, usually bacteria. Often it happens in the context of a pneumonia, injury, or chest surgery. It is one of the various kinds of pleural effusion. There are three ...
,
and
chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD) is a type of progressive lung disease characterized by long-term respiratory symptoms and airflow limitation. The main symptoms include shortness of breath and a cough, which may or may not produce ...
have also been found to be positively affected by DNases properties.
Furthermore, recent studies show that intrapleural
tissue plasminogen activator
Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein involved in the breakdown of blood clots. It is a serine protease () found on endothelial cells, the cells that line the blood vessels. As an enzyme, it catalyzes the conversion ...
(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 in
parapneumonic effusions
A parapneumonic effusion is a type of pleural effusion that arises as a result of a pneumonia, lung abscess, or bronchiectasis. There are three types of parapneumonic effusions: uncomplicated effusions, complicated effusions, and empyema. Uncomp ...
and
empyema
An empyema () is a collection or gathering of pus within a naturally existing anatomical cavity. For example, pleural empyema is empyema of the pleural cavity. It must be differentiated from an abscess, which is a collection of pus in a newly forme ...
.
Other diseases
Sepsis
Sepsis, formerly known as septicemia (septicaemia in British English) or blood poisoning, is a life-threatening condition that arises when the body's response to infection causes injury to its own tissues and organs. This initial stage is follo ...
is a life-threatening
inflammatory disease
Inflammation (from la, inflammatio) is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecu ...
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.
Systemic lupus erythematosus
Lupus, technically known as systemic lupus erythematosus (SLE), is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissue in many parts of the body. Symptoms vary among people and may be mild to severe. Comm ...
(SLE) is an
autoimmune disease
An autoimmune disease is a condition arising from an abnormal immune response to a functioning body part. At least 80 types of autoimmune diseases have been identified, with some evidence suggesting that there may be more than 100 types. Nearly a ...
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 as
apoptotic cells become self-
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
s 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 of
chromatin
Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
. Studies have shown conflicting results on this treatment, however, further research is being conducted to examine the therapeutic benefits of DNase I.
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.
Assays
DNA absorbs
ultraviolet (UV) light with a wavelength of maximal absorbance near 260 nm. This absorption is due to the
pi electrons
In chemistry, pi bonds (π bonds) are covalent chemical bonds, in each of which two lobes of an orbital on one atom overlap with two lobes of an orbital on another atom, and in which this overlap occurs laterally. Each of these atomic orbitals ...
in the
aromatic
In chemistry, aromaticity is a chemical property of cyclic ( ring-shaped), ''typically'' planar (flat) molecular structures with pi bonds in resonance (those containing delocalized electrons) that gives increased stability compared to satur ...
bases of the DNA. In dsDNA, or even regions of
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
where double-stranded structure occurs, the bases are stacked parallel to each other, and the overlap of the base
molecular orbital
In chemistry, a molecular orbital is a mathematical function describing the location and wave-like behavior of an electron in a molecule. This function can be used to calculate chemical and physical properties such as the probability of finding ...
s leads to a decrease in
absorbance
Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative lo ...
of UV light. This phenomenon is called the
hypochromic effect. When DNase liberates
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
s 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 highly
polymerized
In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many for ...
DNA at 25 °C in a 0.1 M NaOAc (pH 5.0) buffer. The unit's name recognizes the Russian-American biochemist
Moses Kunitz, who proposed the standard test in 1946.
A standard enzyme preparation should be run in parallel with an unknown because standardization of DNA preparations and their degree of
polymerization
In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer, monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are ...
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 the
agarose gel
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the t ...
by DNase, which is present in samples punched into the gel.
DNase activity is represented by the size of a dispensed circular well in an agarose gel layer, in which DNA stained by
ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It i ...
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 with
SYBR Green I
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DN ...
or other DNA gel stains.
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.
See also
*
Extracellular DNA (ecDNA)
*
Deoxyribonuclease I (DNase I)
*
Deoxyribonuclease II (DNase II)
*
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...
*
Enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
*
Hydrolysis
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...
*
Phosphodiester linkages
References
External links
Deoxyribonucleasesat the US National Library of Medicine
Medical Subject Headings
Medical Subject Headings (MeSH) is a comprehensive controlled vocabulary for the purpose of indexing journal articles and books in the life sciences. It serves as a thesaurus that facilitates searching. Created and updated by the United States N ...
(MeSH)
Deoxyribonucleases (DNases)from
Thermo Fisher Scientific Inc.
Thermo Fisher Scientific Inc. is an American supplier of scientific instrumentation, reagents and consumables, and software services. Based in Waltham, Massachusetts, Thermo Fisher was formed through the merger of Thermo Electron and Fisher Sci ...
Website
Deoxyribonuclease Ifrom
Sigma-Aldrich
Sigma-Aldrich (formally MilliporeSigma) is an American chemical, life science, and biotechnology company that is owned by the German chemical conglomerate Merck Group.
Sigma-Aldrich was created in 1975 by the merger of Sigma Chemical Company a ...
Solutions website
Deoxyribonuclease I, active sitefrom
InterPro
InterPro is a database of protein families, protein domains and functional sites in which identifiable features found in known proteins can be applied to new protein sequences in order to functionally characterise them.
The contents of InterPro ...
Website
{{Enzymes
EC 3.1.21