Increased Nuclease Activity Research Articles

Nuclease activity of mixed ligand complexes of copper(II) with heteroaromatic derivatives and picoline

New picoline adducts with carbamic acid [(furan-2-yl)methylene]hydrazide–CuII (CFMH) (1); thiocarbamic acid [(furan-2-yl)methylene]hydrazide–CuII (TFMH) (2); carbamic acid [(furan-2-yl)ethylidene]hydrazide–CuII (CFEH) (3), thiocarbamic acid [(furan-2-yl)ethylidene]hydrazide–CuII (TFEH) (4); carbamic acid [(thiophene-2-yl) methylene]hydrazide–CuII (CTMH) (5), thiocarbamic acid [(thiophene-2-yl)methylene]hydrazide–CuII (TTMH) (6), carbamic acid [(thiophene-2-yl)ethylidene]hydrazide–CuII (CTEH) (7), thiocarbamic acid [(thiophene-2-yl)ethylidene]hydrazide–CuII (TTEH) (8) have been prepared and characterized by analytical, i.r., electronic, e.s.r. and c.v. spectral data. The electronic spectra suggest distorted octahedral geometry for all the picoline adducts. E.s.r. g ∥ values lie between 2.251–2.286 at l.n.t. All the adducts undergo a quasi-reversible one-electron reduction in the range +0.47 to +0.51 V versus s.c.e., attributable to the CuIII/CuII redox couple. The electron transfer is much faster in the semicarbazone complexes than in the thiosemicarbazone complexes. All adducts showed increased nuclease activity in the presence of oxidant; the nuclease activity is compared with that of the parent copper(II) complexes.

Nuclease activity of 2-substituted heteroaromatic thiosemicarbazone and semicarbazone copper(II) complexes

Copper(II) Schiff base complexes derived from furan-2-carboxaldehyde, 2-furylmethyl ketone, thiophene-2-carboxaldehyde and methyl-2-thienyl ketone with semicarbazide and thiosemicarbazide have been prepared and characterized by analytical, i.r., electronic, e.s.r. and c.v. spectral data. The electronic spectral d–d band position varies from 744–415 nm in pyridine and 872–371 nm in DMF. E.s.r. g∥ values lie between 2.1439 and 2.3149 at LNT. All complexes undergo quasi-reversible one-electron electrochemical reduction (CuIII/CuII) in the 0.42–0.52 V potential range. The electron transfer is much faster in thiosemicarbazone complexes compared to semicarbazone complexes. All these copper(II) complexes showed increased nuclease activity in presence of oxidant.

Synthesis, spectral studies and nuclease activity of mixed ligand copper(II) complexes of heteroaromatic semicarbazones/thiosemicarbazones and pyridine

Pyridine adducts of copper(II) complexes with thiosemicarbazones and semicarbazones have been synthesized and characterized using elemental analyses, molar conductance, IR spectra, magnetic measurements, electronic and ESR spectra. Copper(II) adducts show quasi-reversible responses (Δ E p=100 mV) in cyclic voltammograms with E 1/2=0.510–0.535 V versus Ag|AgCl at room temperature in N, N-dimethylformamide, attributable to a Cu III/Cu II couple. The spectral studies support the distorted octahedral geometry of these copper(II) adducts. The nucleolytic cleavage activity of all the copper(II) adducts was carried out on double-stranded pBR 322 circular plasmid DNA by using the gel electrophoresis experiment in the presence and absence of oxidant (H 2O 2). In the absence of oxidant a less discernible DNA cleavage was observed, whereas in the presence of oxidant (H 2O 2) all adducts showed increased nuclease activity.

Mutational Analysis of the Hydrolytic Activity of Yeast RNA Polymerase III

For 25 mutant alleles of ret1, encoding the second largest subunit of yeast RNA polymerase III, we have studied the polymerase III nuclease activity, measuring both the total yield and dinucleotide product composition. Mutations affecting amino acids 309-325 gave slightly elevated nuclease activity. In region 367-376, two mutations gave 12-15-fold increased nuclease activity. Our results do not support the catalytic role in nuclease activity proposed for the conserved DDRD motif in this region (Shirai, T., and Go, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9056-9060). Mutations centered on a basic region from amino acids 480 to 490, which aligns with Escherichia coli beta-subunit sequences between Rif(r) clusters I and II, produce changes in the relative yields of A- and G-containing dinucleotides. Four such mutant polymerases pause during elongation at GPy sequences and, in addition, have a reduced frequency of termination at T(5) terminator sequences. We propose that the side chains of these mutationally altered amino acids are in direct contact with bases in the RNA-DNA hybrid very near the growing 3'-end. Two mutations in domain I near the C terminus produced very large increases in exonuclease activity and strongly increased termination, suggesting that this region also contacts the nascent RNA in the hybrid region.

Inhibition of UV-Damaged DNA Recognition Activity in HeLa Cells by Calcium Ionophore A23187: Potential Involvement of Cytosolic Proteins

A UV-damage recognition protein (UVDRP) was detected from HeLa nuclear extracts using an in vitro DNA-binding assay. Treatment of cells with a Ca2+ ionophore (A23187) caused a dramatic inhibition of the UVDRP binding activity from nuclear extracts. In the absence of EDTA, addition of cytosolic extracts to the binding reaction effectively blocked the UVDRP binding activity, associated with an increased nuclease activity. However, pretreatment of cytosolic extracts with proteinase destroyed a majority of the inhibitory effect on UVDRP binding activity. Using extracts from A23187-treated cells, a similar conclusion was drawn except that the inhibitory effect of cytosolic extracts on UVDRP binding was completely diminished by proteinase K. In addition, inclusion of the cytosolic extracts in the binding reaction did not alter the UVDRP binding dissociation pattern by specific competitors. These results suggest the potential involvement of cytosolic proteins, probably through a "all-or-none" action mechanism, in the recognition of UV-damaged DNA of HeLa cells.

Increase in deoxyribonuclease activity in uraemic lymphocytes is caused by the cleavage of the largest polymerase I subunit

Deoxyribonucleases and DNA-dependent RNA polymerase activities in T and B lymphocytes isolated from patients with chronic renal failure and control subjects were studied. The data clearly shows that the nuclease activity in T and B cells isolated from uraemic patients is remarkably enhanced when compared to the control cells. Concomitant with the enhancement in enzyme activity, the reduction in RNA polymerase I activity and quantity was observed. It was found that the increase in nuclease activity and quantity was limited to the group of relatively small nucleases with molecular weights ranging from 14 kDa to 18 kDa. It has been reported previously that these nucleases are among the cleavage products of the largest subunit of DNA-dependent RNA polymerase I. Thus we suggest that the depressed metabolic activity is a characteristic feature of the uraemic lymphocyte cells and the observed increased in DNase activity in those cells is a result of polymerase I degradation.

Single-strand-preferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated

Single-strand-preferring nucleases (EC 3.1.30.1) selectively cleave internucleotide bonds in single-stranded regions of predominantly duplex DNA and DNA.RNA hybrids and extensively degrade denatured DNA and RNA. The functions of single-strand-preferring nuclease in plants are unknown. We have monitored this nuclease activity in flag leaves of wheat (Triticum aestivum L. cv. Chinese Spring) undergoing natural senescence and in primary leaves of wheat seedlings undergoing dark-induced senescence. In falg leaves, nuclease activity remained at basal levels during the first 2 weeks after anthesis, while chlorophyll content increased to a maximum. Nuclease activity then rose in concert with a decline in chlorophyll, reaching a 16-fold elevation at 5 weeks post-anthesis, when 53% of the chlorophyll had been lost. When 8-day-old wheat seedlings were induced to senesce by placing them in darkness, nuclease activity rose without apparent lag, reaching a 13-fold elevation in 7 days, when 61% of the chlorophyll had been lost. The increase in nuclease activity was reversible upon reexposure of seedlings to light, a decline beginning without apparent lag. Reversibility was complete for plants that had been held in darkness for 5 days, with activity returning to the control level in 2 days. These senescence-related changes in nuclease activity, measured in conventional assays, were consistent with concomitant analysis by activity staining of sodium dodecyl sulfate/polyacrylamide gels. We conclude that an increase in single-strand-preferring nuclease activity is closely associated with wheat leaf senescence and that nuclease activity is subject to negative photoregulation.

Single-stranded deoxyribonucleic acid nuclease induced by African swine fever virus and associated to the virion

Infection of Vero cells with African swine fever (ASF) virus resulted in a marked increase of DNase active on single-stranded DNA (ss-DNase). No increase was observed for double-stranded DNA-specific nuclease activity. In contrast to uninfected cell ss-DNase, which has a pH optimum at pH range 8.5–9, virus-induced ss-DNase is most active at pH 7. Differences in sensitivity to several ions and other modifications of the reaction mixture and considerable difference in reaction kinetics suggest that the increase in nuclease activity is due to a new virus-induced enzyme. This is strengthened by the fact that anti-ASF virus antiserum inhibits the activity of ss-DNase from infected cells but not from uninfected cells. Exclusion chromatography of the digests shows that virus-specific ss-DNase is exclusively or predominantly an endonuclease. The increase in nuclease activity of infected cells is proportional to the multiplicity of infection. Virus-specific ss-DNase is synthesized at late times after infection and its synthesis is dependent on viral DNA replication since it is not induced when infected cells are treated with cytosine arabinoside. Most of ss-DNase activity in infected cells is associated to an insoluble cytoplasmic fraction, presumably virosomes. The enzyme can also be detected in partially stripped purified virions which hydrolyze 6.9 ng DNA per microgram viral protein.

The release of nucleases from tobacco pollen

Sugar-unspecific nucleases diffuse out of intact and ungerminated pollen grains of Nicotiana tabacum within 2 min of suspension in pollen medium. No further increase in nuclease activity of the medium was detected during the subsequent pollen cultivation. Two fractions of the nuclease activity are separable by diethylaminoethyl-cellulose (DEAE-C) chromatography differing in relative activities towards native DNA, denatured DNA and RNA. The pH optimum for hydrolysis of the different substrates range from 5.0 to 5.7.

Correlation of increased nuclease activity with enhanced virus reactivation

An increase in nuctease activity, which degraded both unirradiated and ultraviolet (UV)-irradiated DNA, was observed in the extract of monkey Vero cells after irradiation with an appropriate amount of UV. In contrast, no increase was observed with mouse L cells. Neither DNA polymerases nor uracil-DNA glycosylase was enhanced but rather suppressed by UV irradiation in both cell lines. Cytological studies showed that, in Vero cells, the reactivation of UV-irradiated herpes simplex virus was markedly enhanced by irradiating cells with UV before infection. However, no enhancement was observed with L cells. These results suggest that an increase in nuclease activity may be one of underlying mechanisms for the enhanced reactivation of DNA viruses.

Increase in nuclease activity as a possible means for detecting tumor cell sensitivity to anticancer agents.

Histochemical activity of nucleases was investigated in sensitive and resistant rat tumors at several periods after a single dose of cyclophosphamide and was compared with that in a control group of untreated tumors. Intense increase of nuclease activity(mainly that of acid DNAse) appeared only in the cells of the sensitive tumors at 12 hours, reaching its maximum three days after drug administration. Untreated malignant tumors usually have variable amounts of spontaneous necrosis with high nuclease activity. Not the absolute values of high nuclease activity but only the increase of such activity from the basal level of untreated tumors to that measured at frequent periods after trial treatment may be suggested as an early marker of therapeutically induced tumor cell necrosis. However, more investigation is needed before such a marker might be applied as a test for tumor sensitivity to therapeutic agents.

Serum deoxyribonucleases in patients with breast cancer

Serum acid and alkaline DNase levels were determined in the serum of 224 breast cancer patients and 110 healthy individuals. Values above 400 U/ml serum for the acid DNase and 200 U/ml serum for the alkaline DNase were taken as abnormal. Whereas a percentage of breast cancer patients showed increased DNase levels none of the healthy individuals reached an abnormal value. DNase levels were correlated with the clinical status of the breast cancer patients. The percentage of patients with elevated DNase levels was found to be increased with increasing clinical stage. For the acid DNase it was found to be 4 63 ( 6%), 2 11 ( 18%), 7 25 ( 28%), 39 80 ( 49%) and 43 45 ( 96%) for benign tumors and stages I, II, III and IV breast carcinomas, respectively, whereas for alkaline DNase it was 2 63 ( 3%), 2 11 ( 18%), 8 25 ( 32%), 42 80 ( 52%) and 42 45 ( 93%). All 20 of the clinical stage III patients examined 1 month after modified radical mastectomy, as well as 35 other breast cancer patients on adjuvant therapy, had a normal value of DNase levels. With few exceptions i.e., rheumatic fever and polymyositis, no significant increase in nuclease activity was found in the serum of patients with some other conditions causing inflammatory responses and cell death. These preliminary findings indicate that measurements of serum DNase could be used in diagnosis and in monitoring the response to surgical treatment of breast cancer patients.

Increased nuclease activity in cells treated with pppA2'p5'A2'p5' A.

A series of 2'-5'-linked oligo(adenylic acid) triphosphate (2'-5' A) inhibitors of protein synthesis were described recently. These inhibitors are synthesized from ATP by an enzyme activated in interferon-treated cell extracts or rabbit reticulocyte lysates by double-stranded RNA. We show here that 2'-5' A is a potent inhibitor of protein synthesis in intact cells of different origin (human, monkey, hamster, and mouse). At a concentration of 10 nM (in AMP equivalents), protein synthesis is inhibited by 50-85%. There is also a secondary effect on the total RNA synthesis which becomes evident several hours after inhibition of protein synthesis. All of these effects, however, are transient and, after a recovery period, both RNA and protein synthesis resume rates comparable to the appropriate controls. A nuclease activity is detected in cells after treatment with 2'-5'A. The total polyadenylylated RNA is much reduced in comparison to that from untreated cells, and electrophoretic analysis in polyacrylamide slab gels provides evidence for its degradation. Similarly, there is an apparent degradation of ribosomal RNA. Consistent with these results, extracts from cells that had been treated with 2'-5'A manifest an enhanced nuclease activity in vitro on incubation with exogenous RNA. Here, we propose that, as in cell-free systems, the mechanism of action of 2'-5'A in intact cells involves activation of a nuclease. This activation is transient, but the nuclease remains sensitive to further activation by the inhibitor.

Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms.

The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.

Control of the activity of intracellular nucleases in Neurospora crassa.

At least four species of nucleases (nuclease N1, N2, N3 and N4) and one ribonuclease (ribonuclease N3) were detected in extract of wild type mycelia grown in high phosphate media by gel filtration of 0–65% ammonium sulfate precipitate through Sephadex G-100. Nuclease N4 eluted the first is a latent nuclease, the activity of which is not detectable within a week after preparation of the extract but a significant increase in nuclease activity was observed during additional one or two weeks by standing the fraction at 4°C. Nuclease N1 eluted the second is very labile and nuclease N2 eluted the third is stable at the temperature. Nuclease N3 eluted the last was activated within two or three weeks at 4°C. Although all the four nucleases were detected independent of the concentration of orthophosphate in culture media, significantly large amounts of latent ribonuclease (ribonuclease N3) and a number of nucleases including at least one latent nuclease were observed in wild type mycelia grown in low phosphate media. Ribonuclease N3 was determined to be a repressible enzyme. The activities of these constitutive latent nucleases, ribonuclease N3 and a number of nucleases specifically present in wild type mycelia grown in low phosphate media were not observed or significantly reduced in both nuc-1 and nuc-2 mutants, which were deficient to derepress at least eight orthophosphate repressible enzymes relating to phosphate metabolism. A revertant from nuc-2 restored the ability to show activation of at least one of the constitutive latent nucleases.

Dislodgement and thymineless elimination of N-group plasmids

SUMMARYThymineless strains ofEscherichia coliC600 were constructred harbouring both an R factor of the N incompatibility group (R46 or R447b) and a compatible plasmid (Plac-of the A-C group or the Iα plasmid R62), which contained a segment of N group DNA. Selection was made for the transferred plasmid and dislodgement phenomena were manifest either as loss of an entire plasmid or as deletions of a region of plasmid DNA. Even after the two R factors had become established as separate replicons, the N group R factor but not the other plasmid exhibited instability.Thymine starvation of strain C600thy(R447b/R62) increased the elimination rate of the N group plasmid R447b but no elimination of R62 was observed. However, thymine starvation of strain C600thy(R46/Plac-) not only increased the rate of elimination of R46 but also increased the rate of loss of Plac-. There was no detectable increase in nuclease activity in unstarved R46/Plac-strains and it is concluded that dislodgement of R46 from these strains is not due to induction of the nuclease that has been proposed to be responsible for the elimination of N group plasmids during thymine starvation.Two variants of Plac-were isolated. These did not dislodge R46 from unstarved R46/Plac-strains and were not lost during thymine starvation even though thymineless elimination of R46 occurred at normal frequency.

Replication of equine herpesvirus type I: Resistance to hydroxyurea

Hydroxyurea (HU) at concentrations that rapidly and completely inhibit mammalian cellular DNA synthesis does not prevent either equine herpesvirus type 1 (EHV-1) DNA synthesis or virus replication. Analysis by CsCl isopycnic centrifugation of DNA synthesized in HU treated, EHV-1 infected L-M cell cultures demonstrated the synthesis of only viral DNA, whereas both cellular and viral DNA were synthesized in uninhibited, infected cultures. With regard to the mechanism of HU-resistance of EHV-1 DNA synthesis, these studies showed the following: (1) Selective degradation of cellular DNA and increased nuclease activity to provide deoxyribonucleotides for viral DNA synthesis were not induced by infection and/or the inhibitor, (2) HU did not selectively inhibit cellular DNA polymerase activity as both cellular and viral DNA polymerase activities were present in HU-treated, infected cells and were not inhibited by HU added to the in vitro enzyme assay, (3) Cycloheximide inhibition of protein synthesis revealed the requirement of a protein(s) other than viral polymerase at 4 hr postinfection for viral DNA synthesis in the presence of HU. These results do not rule out the possibility that a HU-resistant, viral-induced ribonucleotide reductase activity may be responsible for the HU-resistance of this herpesvirus.

The level of a relatively purine-specific ribonuclease increases in virus-infected hypersensitive or mechanically injured tobacco leaves

The increase in nuclease activity in ‘Xanthi’ tobacco leaves infected with tobacco mosaic virus (TMV) was found to be due mainly, if not entirely, to an increase in the level of a relatively purine (guanine) specific endoribonuclease. The level of the same nuclease increased in the leaf tissues upon mechanical injury. The level of other nucleases contained by the tobacco leaf remained unaltered both upon virus infection and mechanical injury. No correlation between virus multiplication per se and the increase in the level of the relatively purine specific ribonuclease was found.

Source of the nonlinear dependence of bacteriophage SP82 transfection on deoxyribonucleic acid concentration.

Extracts of competent cells of Bacillus subtilis exhibited nuclease activity on radioactively labeled cell deoxyribonucleic acid (DNA). The activity was not decreased when Mg was omitted from the reaction mixture but was decreased to zero by addition of ethylenediaminetetraacetic acid. Of the other metals tested, only Ca increased nuclease activity more than Mg. Addition of 1.5 x 10(-3)m Ca to transfection mixtures increased the nonlinearity of the relationship between number of transfectants and DNA concentration. The inferred role of the Ca-activated nuclease was checked by showing that the ultraviolet enhancement of transfection is reversed by addition of Ca. It was also shown, by testing in three different laboratories, that the Ca ion in water is the likely source of the different nonlinear relationships found in different laboratories.

A study of the deoxyribonucleases and deoxyribonucleic acid polymerase of Escherichia coli K12S after infection with the bacteriopahge T 4r

T 4r infection of Escherichia coli K12S leads to a 4-fold rise in both DNAase and DNA polymerase (deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) activities after 10 min. Part of the increase in nuclease activity seems to be due to the formation of an exonuclease chromatographically associated with DNA polymerase. The new exonuclease associated with λ phage formation in K12S is not seen after T 4r infection of K12S. A large part of the DNA polymerase activity in normal or T 4r-infected cells is attached to ribosomes when extracts are prepared in the presence of 0.01 M Mg 2+. In normal cells the polymerase activity is released from the ribosomes in the absence of Mg 2+. In contrast, in T 4r-infected cells, ribosomal particles prepared in the absence of Mg 2+ still contain significant amounts of DNA polymerase activity.