Dnase

Definition Deoxy Ribonuclease - DNASe production, Mammalian cell lines, Vector Types, method of production, uses- Cystic Fibrosis, Tissue Plasminogen Activator, Bread Making, Applications

1. DNASE M.Meenakshi Assistant Professor, Department of Microbiology, Sri Ramakrishna college of Arts & Science for women Coimbatore

2. Deoxyribonuclease A  deoxyribonuclease  ( DNase , for short) is an  enzyme  that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. Deoxyribonucleases are one type of nuclease, a generic term for enzymes capable of hydrolyzing phosphodiester bonds that link nucleotides. A wide variety of deoxyribonucleases are known, which differ in their  substrate specificities , chemical mechanisms, and biological functions.

3. Modes of action Some DNases cut, or "cleave", only residues at the ends of DNA molecules (exodeoxyribonucleases, a type of exonuclease). Others cleave anywhere along the chain ( endodeoxyribonucleases , a subset of endonucleases ). Some are fairly indiscriminate about the DNA sequence at which they cut, while others, including restriction enzymes, are very sequence-specific . Some cleave only double-stranded DNA; others are specific for single-stranded molecules; and still others are active toward both . DNase enzymes can be inhaled using a nebuliser by cystic fibrosis sufferers. DNase enzymes help because white 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.

4. Types The two main types of DNase found in  metazoans are known as  deoxyribonuclease I  and  deoxyribonuclease II . Other types of DNase include  micrococcal nuclease . Micrococcal nuclease  ( EC   3.1.31.1 ,  S7  Nuclease ,  MNase ,  spleen endonuclease ,  thermonuclease ,  nuclease T ,  micrococcal endonuclease ,  nuclease T' ,  staphylococcal nuclease ,  spleen phosphodiesterase ,  Staphylococcus aureus nuclease ,  Staphylococcus aureus nuclease B ,  ribonucleate ( deoxynucleate ) 3'-nucleotidohydrolase ) is an  endo –exonuclease that preferentially digests  single-stranded nucleic acids. The rate of cleavage is 30 times greater at the 5' side of A or T than at G or C and results in the production of mononucleotides and oligonucleotides with terminal 3'-phosphates. The enzyme is also active against  double-stranded DNA and RNA and all sequences will be ultimately cleave DNase  II is the predominant  DNase  located in lysosomes of cells in various tissues including macrophages (Evans & Aguilera, 2003; Yasuda et al., 1998). With its lysosomal localization and ubiquitous tissue distribution, this enzyme plays a pivotal role in the degradation of exogenous DNA encountered by endocytosis.

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6. immunity Deoxyribonuclease I  (DNase I, encoded by  DNASE1 ) is a specific   endonuclease  facilitating  chromatin  breakdown during  apoptosis .  DNase  I activity is important to prevent  immune stimulation , and reduced activity may result in an increased risk for production of  antinucleosome antibodies ,

7. producing recombinant human DNase I ( a) culturing recombinant human DNase I-producing mammalian cells in a serum-free medium such that the recombinant human DNase I secretes in the medium ; (b) collecting culture supernatant by removing the cells from the culture obtained by the culturing; ( c) subjecting the culture supernatant to an anion-exchange column chromatography to collect a fraction containing the recombinant human DNase I ; (d) subjecting the fraction to a column chromatography that employs a solid phase comprising a material having affinity for a phosphate group to collect a fraction containing the recombinant human DNase I ; (e) subjecting the fraction collected in (d) to a cation-exchange column chromatography to collect a fraction containing the recombinant human DNase I; and (f) subjecting the fraction collected in (e) to a dye affinity column chromatography to collect a fraction containing the recombinant human DNase I.

8. Vector system The method according to claim 1, wherein the recombinant human DNase I-producing mammalian cells are introduced with an expression vector comprising a human elongation factor-1α promoter, a gene encoding rhDNase I downstream thereof, an internal ribosome entry site derived from 5′ untranslated region of murine encephalomyelitis virus further downstream thereof, and a gene encoding a glutamine synthetase still further downstream thereof, and additionally a puromycin or neomycin resistance gene downstream of another gene expression regulatory site .

9. USES DNA REMOVAL IN BIOPROCESSING APPLICATIONS- DNase is commonly used when purifying proteins that are extracted from prokaryotic organisms. Protein extraction often involves degradation of the cell wall. It is common for the degraded and fragile cell wall to be accidentally 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.The DNA is hydrolyzed but the proteins are unaffected and the extract can undergo further purification . DNASE FOOT PRINTING A   DNase footprinting assay [1]  is a  DNA footprinting  technique from  molecular biology / biochemistry  that detects  DNA - protein  interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme,  deoxyribonuclease  (DNase, for short), to cut the radioactively end- labeled DNA, followed by  gel electrophoresis  to detect the resulting cleavage pattern .

10. USES TREATMENT OF DISEASES Tissue plasminogen activator - Intrapleural   tissue plasminogen activator  ( tPA ) combined with deoxyribonuclease has been shown to increase pleural drainage, decrease hospital length of stay, and decrease need for surgery in  parapneumonic effusions  and  empyema . CHRONIC BRONCHITIS. DNase is delivered to the patient in  aerosol  form, and it improves lung function by breaking up the thick mucus in the lungs. The patient is then able to clear the lungs by coughing. Considered among the ten most important advances of 1993—the year the drug was approved by the  Food and Drug Administration  (FDA)—it is also being considered for the treatment of chronic bronchitis. CYSTIC FIBROSIS Scientists discovered that the mucus (slimy secretion) of CF patients is full of DNA, which spills out of white blood cells as they die. DNase is an enzyme (a protein-like substance) that cuts the DNA present in the mucus. At first DNase was made from cows, but many patients had allergic reactions to it. Then a company separated the gene for human deoxyribonuclease , which chops up the protein but does not cause allergic reactions.

11. USES… ACTIN BINDING DNase I binds to the  cytoskeletal  protein  actin . It binds actin monomers with very high (sub- nanomolar )  affinity  and actin polymers with lower affinity. The function of this interaction is unclear. However, since actin-bound DNase I is enzymatically inactive, the DNase- actin  complex might be a storage form of DNase I that prevents damage of the genetic information INDUSTRIAL APPLICATION Enzymes catalyze three main reactions in bread-making: breaking starch into maltose, a complex sugar; breaking complex sugars into simple sugars; and breaking protein chains. 

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14. Assay DNA absorbs UV light with a wavelength of maximal absorbance near 260 nm. This absorption is due to the pi electrons in the aromatic bases of the DNA. In dsDNA, or even regions of RNA where double-stranded structure occurs, the bases are stacked parallel to each other, and the overlap of the base molecular orbitals leads to a decrease in absorbance of UV light. This phenomenon is called the hypochromic effect. When DNAse liberates nucleotides 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 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 DNA at 25 °C in a 0.1 M NaOAc (pH 5.0) buffer. The unit's name recognizes the Russian-American biochemist M. 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 solution is not possible.