DNases activity

ISG20 IFN-stimulated gene product of 20kDA A member of the DEDD exonuclease superfamily with RNAse and DNAse activity... 

CC - - FUNCTION THIS ENZYME HAS BOTH RNASE AND DNASE ACTIV-CC ITY (BY SIMILARITY). 

Some workers prefer to carry out the antibody incubations at 4°C because some monoclonal preparations were found to have DNase activity from mycoplasma contaminants. This should not be a problem with commercial preparations. 

The rate of hydrolysis of DNA, RNA, and polynucleotides can be measured by a sensitive spectrophotometric assay which is based on the hyperchromicity that occurs upon hydrolysis of these substrates (S). The enzyme has a 7-fold greater affinity for denatured DNA than for RNA (8). No inhibitory products accumulate during the course of the reaction. The pH optimum for RNase and DNase activities is between 9 and 10, depending on the Ca2+ concentration. At higher pH values less Ca2+ is required. The inhibitory effect of high Ca2+ observed consistently by many investigators is more pronounced at higher pH values (S). 

Microbial RNases with known substrate specificity are listed in Table XII. Nucleases with DNase activity are not included. It should be noted here that the lists of RNases in both animal and plant kingdoms are presented in the monograph by Privat de Garilhe (4) and in a chapter by E. A. Barnard in Annual Review of Biochemistry (1969) (135). 

The existence of a deoxyribonuclease in E. coli bound to an inhibitory RNA was first suggested by Kozloff (3< ) who found that the DNase activity of freshly prepared extracts could be markedly enhanced by pretreatment with ribonuclease. The enzyme was subsequently purified and freed of inhibitor (39). The purified enzyme termed endonuclease I could, in turn, be competitively inhibited by a variety of RNA s including transfer RNA, and Ri values as low as 10-8 M (nucleotide) have been observed (40). Examination of various purified RNA species and synthetic polyribonucleotides for their inhibitory activity has led... 

In 1964, Tsuda and Strauss discovered a DNase activity in crude extracts of Micrococcus lysodeikticus (later renamed Micrococcus luteus) which required a nucleoside di- or triphosphate for activity (48). This enzyme has recently been purified extensively (2400-fold) and examined in detail by Takagi and his colleagues (49). It has an alkaline pH... 

When acid DNase activity is assayed by the acid-solubility method the optimal DNA concentration is 0.4 mg/ml (9) and higher substrate concentrations appear to be inhibitory (16, 21, 34)- It has been shown, however, that this inhibition is because increasing substrate concentration decreases the efficiency of acid-soluble oligonucleotide release since the number of breaks per unit length of DNA is lower. If a direct method of estimating enzymic activity is used, such as the determination of phosphatase-sensitive phosphate, it can be shown that the inhibition by high substrate seen by the acid solubility method is only apparent (34)-... 

The effect of pH and ions on acid DNase activity has been investigated in several laboratories, and rather different results have been reported. It appears now that many discrepancies result from a rather poor understanding of the complexity of pH and ion effects. In fact, it has been shown (34) that electrolytes and pH modify the acid DNase activity not only by affecting the enzyme itself but also by stabilizing or destabilizing the secondary structure of native DNA. Since the enzyme has a quite different affinity for the native vs. the denatured structure... 

The enzyme levels in the different tissues examined by Cordonnier and Bernardi 35) were found to vary by as much as three orders of magnitude. The highest acid DNase levels were found in lymphatic and tumoral tissues the lowest were found in cells (sperms and erythrocytes) that do not reproduce themselves anymore. This relationship between levels of acid DNase activity and capacity for proliferation or regeneration of a given tissue had already been observed by Allfrey and Mirsky 68). 

Fig. 3. Sephadex G-100 chromatography on DNase I, inhibitor II, and mixture containing the two proteins. (A) DNase I only, (B) inhibitor II only, (C) and (D) both components with different molar excess of inhibitor, (E) equimolar amounts of inhibitor and enzyme, (F) and (G) both components with a different excess of enzyme. Absorbance at 215 nm (solid line) was measured after 20-fold dilution with water using a similarly diluted blank of the elution buffer (0.5 M potassium phosphate, pH 7.6). Each chromatogram was analyzed for DNase activity ( ), inhibitor activity (O), and for the presence of DNase-inhibitor complex, in this figure represented as DNase I activity which was measured on samples of the fractions after adjustment of the pH to 3.5 with HC1 (V). [From Lindberg (34). Copyright 1967 by the American Chemical Society. Reprinted by permission of the copyright owner.]...

The DNase activity of o-phenanthroline-Cu2+ complex bound to DNA in the presence of H202 is also triggered by thiols (Chap. 12). 

Various methods have been developed for the determination of DNase activity. A first group is based on the typerchromicity" method of Kiuiitz and determines... 

M. Tfckahashi and C. Ling. Use of a fluorescent DNA analog for the fluorimettic detection of DNase activity. Anal. Biochent. it 246-249 (1991). 

Usually the linear phase of the absorbtion increase is about 30 sec, and the slope is directly proportional to the enzyme activity. Addition of G-actin leads to an inhibition of DNAse activity. Applying defined concentrations of purified skeletal muscle a-actin gives a standard curve in which ag of actin is proportional to percentage inhibition. When the cell lysates are tested in this system, the determined % of inhibition gives via the standard curve [xg of actin. 25 (xl of cell extract is mixed with 25 (xl DNAse I solution, and then 1 ml of DNA solution is added followed by measurement of the absorbtion. 

Histochemical studies of RNase and DNase activities in normal and... 

Stock solution of RNase Dissolve 2 mg of DNase-free RNase A (Sigma Chemical Co., St Louis, MO) in 1 mL of distilled water. If DNase-free RNase is unavailable, DNase activity is destroyed by boiling the stock RNase solution for 5 min. Aliquots can be stored at -20°C. 

Sigman, D. S., and A. Spassky. DNAse activity of 1,10-phenanthroline-copper ion. Nucl. Acids Mol. Biol. 3 (1989), 13-27. 

Fig, 3. (a) Elution profile of TX-REC when applied to HiTrap Heparin affinity column. The firac-tions containing Tk-REC are indicated, (b) Coomassie Brilliant Blue stained SDS-PAGE of the 10 consecutive fractions containing purified recombinant Tt-REC eluted from the HiTrap Heparin affinity column. Lane M, molecular mass standards lanes 1-10, purified recombinant Tk-REC from fractions 1 to 10, (c) DNase activity of purified recombinant Tk-REC from fractions 1 to 10. Lane C, pUC19 (2 xg) without addition of Tfe-REC lanes 1-10, pUC19 (2 p,g) with Tk-REC (10 p.1) of fractions 1 to 10. Arrows indicate nicked DNA with slow migration rates. 

---