Multiple forms of deoxyribonuclease I

Summary This article will review recent progress on the purification of DNase I (E.C.3.1.4.5) from various sources and the characterization of multiple forms of the enzyme. The chemical basis of the multiple forms in bovine pancreas will be discussed in detail, while for other DNases, including those in ovine pancreas, bovine, mouse and rat parotid, and malt, only the evidence for multiplicity will be presented.     

The inhibition of pancreas deoxyribonuclease

The action of inhibitors on the enzymic degradation of deoxyribonucleic acid and deoxyribonucleohistone by pancreatic deoxyribonuclease has been studied. The results show that the inhibitors may be divided broadly into two classes, namely those which remove the activating magnesium ion, and those which directly interact with the enzyme protein. Active inhibitors prevent both stages (i.e. depolymerisation and hydrolysis) of the degradation of the deoxyribonucleic acid.The products which result from the reaction of nitrogen mustards with deoxyribonucleohistone are partially resistant to the action of deoxyribonuclease. Pre-treatment of deoxyribonucleohistone with other mitotic inhibitors has no effect upon its subsequent degradation with deoxyribonuclease.     

Human genetically polymorphic deoxyribonuclease: purification, characterization, and multiplicity of urine deoxyribonuclease I

A deoxyribonuclease I was purified from the urine of a 46-year-old male (a single individual) by using a series of column chromatographies to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to be a glycoprotein, containing 1 fucose, 7 galactose, 10 mannose, 6 glucosamine, and 2 sialic acid residues per molecule. The N-terminal amino acid sequence up to the 27th residue of the enzyme was similar to that of pancreatic deoxyribonuclease I from bovine and other species. The catalytic properties of the enzyme derived from a single individual closely resembled those of deoxyribonuclease I purified from human urine collected from several volunteers [Ito, K. et al. (1984) J. Biochem. 95, 1399-1406]. The purified enzyme was found to consist of multiple forms with different pI values. These findings are compatible with the existence of genetic polymorphism of deoxyribonuclease I in human urine previously reported [Kishi, K. et al. (1989) Hum. Genet. 81, 295-297]. This multiplicity of the urine enzyme might be due to variations in the primary structure and/or differences in the content of sialic acid.     

Digestion of chromatin with deoxyribonuclease II

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded. Early in the degradation process DNA was converted into double stranded fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. After denaturation DNA from digested chromatin was resolved into discrete single stranded fractions, exact multiples of a ten-nucleotide length, forming a pattern very similar to that observed with DNAse I.     

Role of deoxyribonuclease in genetic transformation

It was found that an inhibition of the relatively low deoxyribonuclease activity in the highly transformable and stable Pneumococcus R6bd strain results in a decrease of transformation frequency. The opposite is true about the Pneumococcus PMSII57 strain where an inhibition of its high deoxyribonuclease activity results in an increase of transformation frequency. It was observed that ribonucleic acid and especially t-RNA inhibits deoxyribonuclease activity in pneumococcus strains. Hence the kinetics of the effect of t-RNA as a function of time was examined with respect to the frequency of transformants in recipient cells of R6bd strain. It was found that t-RNA inhibits transformation. Maximum inhibition occurs if t-RNA is added to the DNA-bacterial complex at the time of penetration of transforming DNA into recipient cells. The results indicate that in a highly transformable and stable Pneumococcus strain the penetration of DNA is associated with the regulation of optimal deoxyribonuclease activity.     

Role of deoxyribonuclease in genetic transformation

The deoxyribonuclease activity was investigated in cell-free filtrates of cultures of strains of different transformability. The highly transformable stable strains display a relatively low, probably optimal, deoxyribonuclease activity. Transformable but unstable strains produce a very high deoxyribonuclease activity in comparison with the aforementioned ones. In some nontransformable strains no deoxyribonuclease activity could be detected. The role of deoxyribonuclease in the competence of recipient cells s discussed.     

Electrochemical assay for deoxyribonuclease I activity

A thiolated oligonucleotide having three ferrocenes was immobilized on a gold electrode through the sulfur-gold linkage. This electrode showed a current response based on the redox reaction of the ferrocene moieties and this response was decreased after treatment with deoxyribonuclease I (DNase I), suggesting the disappearance of the ferrocene moieties on the electrode by the DNase I digestion. A linear correlation between i₀ and i, which are current peaks before and after DNase I treatment, respectively, was observed and this slope was decreased with increase in the amount of DNase I. No current decrease was observed in the presence of EDTA or RNase A instead of DNase I. These results suggested that the current decrease responded specifically to the amount of DNase I and this electrode could be used for an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37 °C for 30 min, a quantitative analysis could be achieved in the range of 10⁻⁴-10⁻² units/μl of DNase I.