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

3. 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.

4. 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.

5. 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.

6. VIDEO

7. 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,

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. APPLICATIONS

14. APPLICATIONS….

15. 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.