DNAases

Deoxyribonuclease (DNAase), an enzyme that degrades deoxyribonucleic acid, has been used in patients with chronic bronchitis, and found to produce favorable responses presumably by degrading the DNA, contributed by cell nuclei, to inflammatory mucus (213). Lysozyme [9001 -63-2] hydrolyzes the mucopeptides of bacterial cell walls. Accordingly, it has been used as an antibacterial agent, usually in combination with standard antibiotics. Topical apphcations are also useful in the debridement of serious bums, cellulitis, and dermal ulceration. 

A suitable method for this was introduced by Caspersson (mid-1920s-ca. 1940) who designed and successfully exploited UV microscopy so that the extent of the absorbtion could be determined quantitatively. Selective hydrolysis by RNAase or DNAase was used so that the DNA content of the cell could be estimated. The procedure still required reproducible preparation of sections to allow light to be transmitted and the enzymes to get access to the nucleic acids. 

Figure 20.35 Mechanisms by which external or internal stress leads to cell damage resulting in apoptosis. The stress leads to activation of initiator proteolytic enzymes (caspases) that initiate activation of effector caspases. These enzymes cause proteolytic damage to the cytoskeleton, plasma membrane and DNA. The activation of DNAases in the nucleus results in cleavage of DNA chains between histones that produces a specific pattern of DNA damage which, upon electrophoresis, gives a specific pattern of DNA fragments. The major endproduct of apoptosis are the apoptolic bodies which are removed by the phagocytes.

DNAase inhibitor ICAD (inhibitor of caspase-activated DNAase) Inhibitor of a DNAase responsible for DNA fragmentation (Enari et al, 1998). 

DNA vaccines are being explored but it has proved to be difficult to deliver the naked DNA to cellular sites where the appropriate antigen will be produced. Gold particles coated with DNA plasmids have been evaluated. Naked DNA is wasteful in some senses because relatively large quantities are required for even small quantities of translation and adjuvants are required. In addition there is no protection against environmental DNAase enzymes. However, DNA and the appropriate protein antigen may have more potential. 

The approval of genetically engineered version of human DNAase, which breaks down protein accumulation in the lungs of CF patients. It was the first new therapeutic drug for the management of cystic fibrosis in over 30 years... 

Endonucleases. Endonucleases provide for DNA cleavage into small ( 200 base pairs) fragments, which is an essential step in apoptotic cascade (Wyllie 1980 Wyllie 1998). Endonucleases are stimulated by Ca2+ and their activation was detected in several cell types undergoing apoptosis (McConkey et al., 1988 Aw et al., 1990). The intimate mechanisms of endonucleases action remain not fully described at least in part they can be explained through the involvement of caspase-3 activated endonuclease (or caspase-activated DNAase - (Enari et al., 1998)). Nonetheless, the chromatin fragmentation was also observed in cell (and caspase)-free system, when isolated nuclei were treated with Ca2+ and ATP, suggesting the existence of caspase-independent DNA cleavage mechanism (Jones et al., 1989). 

Chiriboga, L., Yee, H. and Diem, M. (2000) Infrared spectroscopy of human cells and tissue. Part VII FT-IR microscopy of DNAase- and RNAase-treated normal, cirrhotic, and neoplastic liver tissue. Appl. Spectrosc. 54, 480-5. 

Free 3 -hydroxyl ends may occur naturally in double stranded DNA or as a result of deliberate nicking (i.e. single stranded breakage) of the molecule with endonucleases such as DNAase I (Figs. 1.2. and 1.3.). Such nicks are usually found to be randomly distributed along a duplex DNA. In a linear duplex the presence of nicks has little effect on the physicochemical properties of the molecule and for that reason they are often referred to as hidden breaks. To discover whether a linear duplex DNA is nicked or not... 

Fig. 3.9. Diagram showing the principle of the nick-translation sequencing procedure. For the purpose of illustration the single unique product generated from one nicked molecule is shown. With a heterogeneous set of nicked duplexes nick-translation will proceed from each gap until terminated by the incorporation of a dideoxynucleotide. Since the corresponding deoxynucleotide is also incorporated in competition with the dideoxynucleotide, the eifect of non-random cleavage with DNAase I is minimized.

Fig. 3.13. Diagrammatic representation of the Forward-Backward procedure. A double-stranded DNA fragment [32P] labelled (asterisk) at one 5 -end is represented at the top of the figure. DNA polymerase I and a nucleotide chain inhibitor (e.g. ddA) are added. Contaminating DNAases in the Poll preparation produce nicks, indicated by the vertical arrows. From the 3 -end created by each nick, the reaction catalysed by Poll proceeds in the 5 - to 3 -direction (Forward reaction) provided dNTPs (dG, dT, dC) are present if they are not added the reaction proceeds exonucleolytically in the 3 - to 5 -direction (Backwards). The numbered lines represent the DNA fragments which arise from the similarly numbered DNA nicks. The hypothetical DNA sequence illustrates the complementary results obtained from the Forward and Backward reactions with repeated nucleotides e.g. the sequence AA. In the Forward reaction the proximal A will be represented by a strong band and the distal A by a weak band. The converse is true for the Backward reaction. The dotted lines 4 and 5 signify those reactions which proceed 5 - 3 (Forward) in the Backwards procedure.

In this experiment the Pstl fragment was first digested with DNAase II in sodium acetate buffer, pH 4.7 at room temperature and the reaction halted by chilling and extraction with phenol. After precipitation the DNA was electrophoresed on an 8% polyacrylamide gel and a slice of gel, corresponding to fragments of chain length 150-250 nucleotides cut out and eluted. The 3 -terminal phosphates were removed by treatment with alkaline phosphatase and, after denaturation and removal of the phosphatase with phenol, the DNA was reprecipitated and dissolved in a small volume of water. 

Fig. 4.9. Outline of a procedure for preparing flush-ended fragments from a DNAase U digest. The products may be ligated with RI linker and cloned into...

A relatively large excess of linker is required to minimize end to end ligation of the DNAase II fragments yielding false sequences. 

In a study on the effect of divalent cations on the mode of action of DNAase I, Campbell and Jackson (1980) found that in the presence of the Mn++ ions DNAase I is able to cut both DNA strands within a duplex at or near the same point. This ability to cut both strands is inhibited at lower temperatures and by the addition of a monovalent ion or another divalent cation which is not a transition metal ion. Transition metal ions thus appear to promote the localized unwinding of duplex DNA into a form where DNAase I can introduce breaks into both strands. These observations therefore suggest another route for producing a more or less random set of fragments which, after limited polishing of the ends with DNA polymerase, could be ligated to the RI linker and subsequently cloned into M13mp2. 

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