Inhibition Of Nuclease Activity Research Articles

Inhibition of nuclease activity by a splice-switching oligonucleotide targeting deoxyribonuclease 1 mRNA prevents apoptosis progression and prolong viability of normal human CD4+ T-lymphocytes

The nuclease activity of deoxyribonuclease 1 (DNase I) is regulated by alternative splicing (AS) of its mRNA. The aim of this study was to define the ability of a splice-switching oligonucleotide (SSO) that base-paired with DNase I pre-mRNA to induce AS and inhibit nuclease activity in human T, B and NK lymphocytes. The SSO for DNase I could significantly downregulate the expression of full-length active DNase I and upregulate a truncated splice variant with a deleted exon 4. Such an induction of AS resulted in inhibition of nuclease activity and slowed apoptosis progression in anti-CD95/FAS stimulated lymphocytes. These results should facilitate further investigations of apoptosis regulation in lymphocytes and demonstrate that SSOs for DNase I are promising cytoprotective agents.

Increased Neutrophil Extracellular Trap–Mediated Staphylococcus aureus Clearance Through Inhibition of Nuclease Activity by Clindamycin and Immunoglobulin

The Gram-positive human pathogen Staphylococcus aureus causes a variety of human diseases such as skin infections, pneumonia, and endocarditis. The micrococcal nuclease Nuc1 is one of the major S. aureus virulence factors and allows the bacterium to avoid neutrophil extracellular trap (NET)–mediated killing. We found that addition of the protein synthesis inhibitor clindamycin to S. aureus LAC cultures decreased nuc1 transcription and subsequently blunted nuclease activity in a molecular beacon–based fluorescence assay. We also observed reduced NET degradation through Nuc1 inhibition translating into increased NET-mediated clearance. Similarly, pooled human immunoglobulin specifically inhibited nuclease activity in a concentration-dependent manner. Inhibition of nuclease activity by clindamycin and immunoglobulin enhanced S. aureus clearance and should be considered in the treatment of S. aureus infections.

Mechanism of DNA cleavage by the DNA/RNA-non-specific Anabaena sp. PCC 7120 endonuclease NucA and its inhibition by NuiA

A structural model of the DNA/RNA non-specific endonuclease NucA from Anabaena sp. PCC7120 that has been obtained on the basis of the three-dimensional structure of the related Serratia nuclease, suggests that the overall architecture of the active site including amino acid residues H124, N155 and E163 (corresponding to H89, N119 and E127 in Serratia nuclease) is similar in both nucleases. Substitution of these residues by alanine leads to a large reduction in activity (<0.1 %), similarly as observed for Serratia nuclease demonstrating that both enzymes share a similar mechanism of catalysis with differences only in detail. NucA is inhibited by its specific polypeptide inhibitor with a K i value in the subpicomolar range, while the related Serratia nuclease at nanomolar concentrations is only inhibited at an approximately 1000-fold molar excess of NuiA. The artificial chromophoric substrate deoxythymidine 3′,5′-bis-( p-nitrophenyl phosphate) is cleaved by NucA as well as by Serratia nuclease. Cleavage of this analogue by NucA, however, is not inhibited by NuiA, suggesting that small molecules gain access to the active site of NucA in the enzyme-inhibitor complex under conditions where cleavage of DNA substrates is completely inhibited. The active site residue E163 seems to be the main target amino acid for inhibition of NucA by NuiA, but R93, R122 and R167 (corresponding to K55, R87, R131 in Serratia nuclease) are also involved in the NucA/NuiA interaction. NuiA deletion mutants show that the structural integrity of the N and C-terminal region of the inhibitor is important for complex formation with NucA and inhibition of nuclease activity. Based on these results a mechanism of DNA cleavage by NucA and its inhibition by NuiA is proposed.

Effects of Soluble Fractions from Untreated and SiO2-treated Subcellular Particles of Macrophages on Nucleic Acid Metabolism in Isolated Nuclei of Experimental Granulation Tissue.

Nuclei isolated from proliferating granulation tissue were incubated with 20 000 g supernatants from untreated and SiO2-treated subcellular particles of rat peritoneal macrophages in the presence of radioactive nucleic acid precursors. The supernatant from SiO2-treated subcellular particles increased the incorporation of [3H]CTP into nuclear RNA maximally by 26% at 5 min, and that of [methyl-3H]dTTP into DNA by 16% at 20 min. The release of radioactivity from labeled DNA was suppressed simultaneously. An RNase preparation from rat peritoneal macrophages enhanced the release of radioactivity from labeled DNA similarly as the soluble fraction from untreated subcellular particles of macrophages. The results suggest that the effects of the soluble fractions upon DNA metabolism of granuloma cells are at least partly independent of the effects on RNA metabolism and that the soluble fraction from SiO2-treated subcellular particles of macrophages stabilizes DNA through inhibition of nuclease activity.

Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity.

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease.

Influence of polyamines on the activity of DNA polymerase I from Escherichia coli

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A—T) n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl 2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA) n · (dT) 10 and (rA) n · (dT) 10 (20 : 1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA) n · (dT) 10 and somewhat less for (dA) n · (dT) 10. The apparent K m for the primer (rA) 10 · (dT) 10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent K m for dTTP. The influence of polyamines on both the 5′ → 3′ and 3′ → 5′ nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A—T) n and T 7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.

Inhibition of nuclease activity in [formula omitted] following infection with bacteriophage SP82G

The total nuclease activity of cell extracts of B. subtilis was characterized and shown to decrease following infection with bacteriophage SP82G. This inhibition was maximal at 6 minutes after infection at 37° and 9 minutes after infection at 33°. The bulk (80%) of the inhibition was chloramphenicol sensitive but a small part (20%) was shown to be chloramphenicol insensitive. Extracts from infected cells were shown to have decreased nuclease activity on a variety of substrates.

Dependence of transfection efficiency of calcium treated Escherichia coli cells on bacterial genotype and form of Lambda DNA.

The transfecting activity of linear lambda DNA is 100 times higher in calcium treated E. coli K12 (lambda i434) than in non-lysogenic strains: the levels of transfection are 1-2.10(7) and 1-2.10(5) infective centers per 1 mug of lambda DNA respectively. The high efficiency of lysogenic cells transfection is not due to the spontaneously liberated "helper" phage. Evidently, it is called forth by transfecting DNA-prophage recombination or/and by inhibition of nuclease activity in lysogenic cells. Both ring forms lambda DNA (supercoiled and open circles) show very low infectivity, if any, in calcinated cells.

Specific inhibition by ATP and other properites of an endonuclease of Neurospora crassa.

An endonuclease specific for single-stranded DNA and RNA was purified from Neurospora crassa conidia by the method of Linn and Lehman (J. Biol. Chem. 240, 1287 (1965)). The activity of the enzyme was measured by following the rate of release of acid-soluble material at 37 °C from either denatured (single-stranded) or native DNA. After an initial lag period, the length of which was inversely proportional to enzyme concentration, the release of acid-soluble material occurred at a rate which was directly proportional to enzyme concentration. Freshly purified enzyme catalyzed the release of acid-soluble material from denatured DNA 50–70 times faster than from native DNA. This ratio of activities increased to approximately 200 following storage at 0–4 °C. Throughout this "ageing" period 2 × 10−4 M ATP inhibited the activity of the enzyme toward both denatured and native DNA by 50%. The following nucleoside phosphates, at a concentration of 4 × 10−4 M, had no effect on the activity of the endonuclease toward denatured DNA: ADP, AMP, ITP, XTP, CTP, dTTP, and UTP. GTP at 4 × 10−4 M inhibited this activity by 15%. Both ATP and dATP at 1 × 10−4 M inhibited endonuclease activity against denatured DNA by about 25%. The inhibition by ATP was noncompetitive over the range of concentration 0.11–0.75 mg DNA per milliliter. Specific inhibition of nuclease activity by ATP has not been previously reported.Several other properties of the endonuclease were examined. There is evidence that endonuclease action was inhibited by accumulation of the products of digestion. The enzyme lost activity toward denatured DNA in a medium containing 0.3 M NaCl but activity was partially restored in the presence of 4 × 10−3 M mercaptoethanol. The endonuclease did not digest DNA–RNA hybrid to any appreciable extent.