Deoxyribonuclease specificity

Restriction endonuclease A deoxyribonuclease which cuts DNA at specific sequences which exhibit twofold symmetry about a point. Name derives from the fact that their presence in a bacterial cell prevents (restricts) the growth of many infecting bacteriophages. 

Patients suffering from cystic fibrosis often use various aerosolized drugs. To reduce the viscosity of the mucus in the airways, recombinant human deoxyribonuclease is used. This enzyme is the first recombinant protein that has been developed for specific delivery to the lungs via the airways. It has a local action on the mucus in the airways and its absorption is minimal. Another drug that decreases the viscosity of the mucus is acetylcysteine. Aerosolized antibiotics are a further group of therapeutics that is widely used by cystic fibrosis patients. Solutions of antibiotics like tobramycin or colistin are used in nebulizers to prevent exacerbation of the disease. Pentamidine has been used for the prophylaxis of Pneumocystis pneumonia in patients infected with HIV virus, while chronic rejection of lung transplants provided a reason to develop an aerosol formulation of cyclosporine A. 

In addition to the enzymes that catalyse the formation of nucleotides and polynucleotides, a large number of catabolic systems exist which operate at all levels of the internucleotide pathways. The ribonucleases and deoxyribonucleases that degrade polynucleotides are probably not significantly involved in purine analogue metabolism, but the enzymes which dephosphorylate nucleoside 5 -monophosphates are known to attack analogue nucleotides and may be of some importance to their in vivo activity. Phosphatases of low specificity are abundant in many tissues [38], particularly the intestine [29]. Purified mammalian 5-nucleotidases hydrolyse only the nucleoside 5 monophosphates [28] and... 

Site-specific deoxyribonuclease (type 11)— restriction endonuclease ... 

Enzymes catalyzing cleavage of DNA, including endo-deoxyribonucleases that generate 5 -phosphomono-esters [EC 3.1.21.x], endodeoxyribonucleases that produce products other than 5 -phosphomonoesters [EC 3.1.22.x], site-specific endodeoxyribonucleases acting on altered bases [EC 3.1.25.x], and exodeoxyribonucleases producing 5 -phosphomonoesters [EC 3.1.11.x]. A few examples are ... 

Type II site-specific deoxyribonuclease [EC 3.1.21.4], also referred to as type II restriction enzyme, catalyzes the endonucleolytic cleavage of DNA to give specific, double-stranded fragments with terminal 5 -phosphates. Magnesium ions are required as cofactors. 

The question of enzyme specificity for irradiated polynucleotides is taken up in more detail in the recent review of Johns.11 The specificities of four enzymes, spleen phosphodiesterase, snake venom phosphodiesterase, pancreatic ribonuclease, and pancreatic deoxyribonuclease are discussed. 

Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme ), and Factor VIII. In the case of rhDNase, Ca2+ ions (up to 100 mM) increased the stability of the enzyme through a specific binding site (64). In fact, the removal of calcium ions from the solution with EGTA caused an increase in deamidation and aggregation. However, this effect was observed only with Ca+2 ions other divalent cations, Mg2+, Mn2+, and Zn2+, were observed to destabilize rhDNase. Similar effects were observed in Factor VIII. Ca2+ and Sr2+ ions stabilized the protein, whereas others such as Mg2+, Mn2+ and Zn2+, Cu2+, and Fe2+ destabilized the enzyme (65). In a separate study with Factor VIII, a significant increase in the aggregation rate was observed in the presence of Al3+ ions (66). The authors note that other excipients like buffer salts are often contaminated with Al3+ ions and illustrate the need to use excipients of appropriate quality in formulated products. 

All of the bacterial deoxyribonucleases that have been examined in detail possess a specificity directed in varying degrees toward the secondary structure of the polydeoxynucleotide. With one recent exception, none of the deoxyribonucleases shows a simple base specificity whereby they attack phosphodiester bonds adjacent to a single base. However, it is now clear that several of the endonucleases may possess an extremely high order of specificity and have the capacity to recognize and attack one or a few phosphodiester bonds in polydeoxynucleotide chains composed of many thousands of internucleotide linkages. 

A deoxyribonuclease not normally present in E. coli is rapidly synthesized after infection with phage T5 (47). This DNase appears at approximately the same time as the other early phage-specific enzymes (DNA polymerase and deoxynucleotide kinase, etc.) induced following infection with this bacteriophage. 

During the past decade a previously accepted notion that the deoxyribonucleic acid (DNA)-deoxyribonuclease (DNase I) reaction runs a uniform course with a uniform specificity began to be seriously doubted. It is now realized that striking differences exist between the early and terminal stages of the same reaction. The observed differences are not limited solely to the rate of the reaction but include variations in endo-or exonucleolytic character, in the effect of the divalent cation, and, finally, in the specificity toward the bases adjacent to the bond that is cleaved. 

The reasons for selecting pancreatic DNase I as one of the two representative of mammalian DNases are to a large extent historical. Deoxyribonuclease I was the first enzyme to be recognized as specific for DNA (18-15), the first DNase to produce 5 -monoesterified products (16, 17), the first DNase to be crystallized (18), the first DNase to have a specific protein inhibitor (19-23), the first DNase shown to produce nicks on one strand in preference to scission of both strands (24, 25). A new first has been added recently (25a) DNase I was covalently coupled to porous glass, thus supplying an insoluble DNase. 

A method for the partial purification of spleen exonuclease was described by Heppel and Hilmoe in 1955 (13) and by Hilmoe in 1960 (14) this was later improved by Razzell and Khorana (15) and Richardson and Kornberg (16). In 1966, we described a novel purification procedure (10) leading to an enzyme preparation with a specific activity comparable to that of the best preparation of Razzell and Khorana (15). Enzyme yields were, however, low the method was therefore modified and satisfactory results were obtained (11). The new method involves the preparation of a crude enzyme obtained essentially as in the case of acid deoxyribonuclease (5, 17). The main differences are that acidification to pH 2.5 is avoided and (NH4)2S04 fractionation is done between 35 and 60%> saturation. The crude enzyme is then purified by chroma-... 

In the small intestine, ribonuclease and deoxyribonuclease I, which are secreted in the pancreatic juice, hydrolyze nucleic acids mainly to oligonucleotides. The oligonucleotides are further hydrolyzed by phosphodiesterases, also secreted by the pancreas, to yield 5 - and 3 -mononucleotides. Most of the mononucleotides are then hydrolyzed to nucleosides by various group-specific nucleotidases or by a variety of nonspecific phosphatases. The resulting nucleosides may be absorbed intact by the intestinal mucosa, or they may un-... 

Nucleases are enzymes that degrade nucleic acids by cleaving phosphodiester linkages. They may be specific for DNA or RNA, or they may act on both. A nuclease specific for DNA is called a deoxyribonuclease (DNase) and for RNA, ribonuclease (RNase). 

For a variety of purposes, procedures have been described to ascertain the nature of the complexes between regulatory proteins and DNA. An early approach was to form a complex between the DNA and the protein and then to subject this complex to digestion with deoxyribonuclease I. The particular region of DNA in specific contact with the protein is protected by the latter from enzymic digestion (see Kadona-... 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10" mM. Phosphate and chloride ions acted as competitive inhibitors. 

Warming may have a beneficial effect. For instance, the temperature of fluids atomized in air-jet nebulizers decreases by approximately 10-15°C during use, resulting in bronchoconstriction in some asthma suffers.Bronchoconstriction, which is most marked at 5°C, disappears at 37°C and thus may be minimized by using an ultrasonic device. Furthermore, when solutions of drugs with low solubility are to be nebulized, ultrasonic nebulizers, which warm the solutions, may be preferable to air-jet devices, which cool them and may cause precipitation. However, the heat generated may harm heat-labile materials such as diethylenetriaminepentoacetic acid ( mTc-/DTPA), " proteins, and some antibiotic solutions. Thus, ultrasonic nebulizers are specifically prohibited for aero-solization of recombinant human deoxyribonuclease (rhDNase). ... 

In the body, enzymes are compartmentalized and function under highly restricted conditions. Some enzymes (e.g., proteinases) are not substrate-specific. When present in active form in an inappropriate part of the body, they act indiscriminately and cause considerable damage to the tissue. Inhibitors inactivate these enzymes at sites where their action is not desired. Proteinase inhibitors, which are themselves proteins, are widely distributed in intracellular and extracellular fluids. Protein inhibitors of enzymes other than proteinases are relatively rare. Such inhibitors are available for a-amylases, deoxyribonuclease I, phospholipase A, and protein kinases. 

A variety of enzymes break phosphodiester bonds in nucleic acids deoxyribonucleases (DNases) cleave DNA and ribonucleases (RNases) cleave RNA. DNases usually are specific for single- or double-stranded DNA although some DNases can cleave both. DNases can act as exonucleases in which they remove one nucleotide at a time from either the 3 or 5 end of the strand. Other DNases function as endonucleases and are specific for cleaving between particular pairs of bases. 

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