Pancreatic DNase

E. Physical Methods and Analytical Techniques.—Nucleotide maps of enzymic digests of DNA have been obtained using the same ionophoretic techniques as have been developed for RNA digests. Pancreatic DNase and Neurospora crassa endonuclease produce very similar maps with E. coli DNA but this technique still awaits the discovery of specific DNases. 

Deoxyribonuclease I [EC 3.1.21.1], also known as pancreatic DNase and thymonuclease, catalyzes the endonucleolytic cleavage of DNA, preferring dsDNA, to a 5 -phosphodinucleotide and 5 -phosphooligonucleotide end products. 

Deoxyribonuclease II [EC 3.1.22.1], also referred to as pancreatic DNase II, catalyzes the endonucleolytic hydrolysis of DNA (preferring double-stranded DNA) to produce 3 -phosphomononucleotide and 3 -phos-phooligonucleotide end products. 

The digestive enzyme, pancreatic DNAse I, makes single-stranded cuts in double-stranded (ds) DNA. 

The haplotomic mechanism is similar to that already known to occur with pancreatic DNase 31, 32) and contributes to the molecular weight decrease only after a time lag, during which single breaks accumulate on the DNA strands. The ratio of total bonds broken to bonds broken by the diplotomic mechanism has been estimated, in different ways, to lie at least initially between 1.5 and 3 33). 

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. 

Fig. 4. Scheme of the preparation containing P-labeled phosphomonoesters at single-strand breaks. The two strands of Ti DNA duplex are schematically represented by two parallel lines and only the 5 termini are designated. After the introduction of single-strand breaks into DNA by incubation with pancreatic DNase, the phosphomonoesters formed are removed by phosphatase at 65°. The 5 termini are then labeled by incubation with polynucleotide kinase. From Weiss el al. (56). 

DNA are readily accessible to RNA polymerase binding and transcription. By contrast, most DNA in eukaryotic cells exists in a condensed form (chromatin), which is not readily accessible to transcription. The small fraction of DNA accessible to the RNA polymerase in any given cell type is especially sensitive to cleavage by mild treatment with bovine pancreatic DNase I. These regions of the DNA often contain bound RNA polymerase, modified histones, and additional nonhistone proteins. Active regions are often undermethylated compared with the total DNA. Most of the methylated groups in DNA are on the C residues in the CG sequence. 

Bovine pancreatic DNase I was purified in 1945 [3], and it was the first DNase to be crystallized [4]. Until the availability of recombinant hrenun DNase (rUDNase) in the late 1980s [5], bovine pancreas remained the principal source of DNase 1, for biochemical as well as for clinical investigations. 

The three-dimensional structure of bovine pancreatic DNase I was first determined st 3,5 A, rssolstics by x- ay tEfTisction from single crystals [23], DNase I proved to be a compact a,fJ-pn tin with two sixstranded sheets packed against each other forming the cove of a sandwich type structure. Tbe... 

Most of the differences between the human and bovine pancreatic DNase I are located at the surface of the molecule in hydrophilic regions, which might be... 

Besides the use of bovine pancreatic DNase I and rhDNase for the treatment of CF, the enzyme was also investigated for the treatment of other respiratory... 

Since 1969, bovine pancreatic DNase 1 has been used for the treatment qf ulcers in dermatology [82-84]. It degrades the polynucleotides that are a main constituent of dwid ullr. One dermatolosical moduet based on DNase f is EImc . 

This ointment contains both bovine pancreatic DNase I as well as a fibrinolytic complex formulated in a mixture of polyethylene glycol and paraffin oil [85]. 

S. Moore. Pancreatic DNase. In Tftr Enzymes, Vol. XIV (P. D Boyer, cd,). Academic Press, New Yctit, iyai, pp. 281-296. 

A. F. Worrell and B. A. Connolly. Hie chemical synthesis of a gene coding for bovine pancreatic DNase I and Us cloning and expression in Escherichia coll. J, BioL Chem. 

A. J. Doherty, A. F. Worrall, and B. A Connolly. Mutagenesis of the DNA binding residues in bovine pancreatic DNase 1 An investigation into ihe mechanism of sequence discrimination by a sequence selective nuclase Nucleic Acids Res. 19 6129-6132 (1991). 

The other major enzymes in pancreatic juice of relevance to our discussion are the nucleases. Human pancreatic DNase I has a molecular weight of around 30 kDa, shows maximal activity in the pH range 7.2-7.6 and requires metal ions such as Mn+, Mg+ or Co+ in the presence of Ca+. It is an endonuclease that hydrolyses the phosphodiester linkages in both single- and double-stranded DNA at multiple sites (Funakoshi et al. 1977 Takeshita et al. 2000). 

Pancreatic DNase (1 mg/ml in sterile IX saline-citrate)— Dissolve 1 mg of pancreatic DNase (Worthington Biochemical Corp., Freehold, N.J.) in 5 ml of sterile IX saline-citrate. Refrigerate or freeze until use. If possible, pass through a sterile bacterial filter (e.g., Millipore, type HAW) into a sterile test tube. Stable for 48 hr. 

DNA polymerase and 5 - 3 exonuclease functions of the enzyme. Nucleotide hydrolysis in the 5 - 3 direction concomitant with nucleotide polymerization results in translocation of the position of the discontinuity by a process termed nick translation (Fig. 2) (30). Discontinuities or nicks in DNA can be introduced into intact DNA by limited digestion with pancreatic DNase I, which generates 3 -hydroxyl termini in double-stranded DNA. If radioactive nucleotides are used in the reaction with DNA polymerase 1, randomly and uniformly labeled DNA is produced (31). 

Experiments in the 1950s and 1960s showed that DNA is present in purulent lung secretions, while it is absent in noninfected secretions [6,7]. As early as 1950, Armstrong reported that bovine pancreatic DNase I significantly reduced the viscosity of purulent lung secretions in vitro [8]. These important... 

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