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PubMedDate not supplied

Human cytomegalovirus terminase as a target for antiviral chemotherapy.

Herpesviral DNA packaging is a complex process involving binding and cleavage of DNA containing the specific DNA-packaging motifs, pac1 and pac2, and packaging of the resulting unit-length genomes into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of human cytomegalovirus the terminase consists of the proteins pUL56 and pUL89. While pUL56 (i) mediates the specific binding to pac sequences on the concatamers, (ii) provides energy for the translocation of the DNA to the procapsids and (iii) associates itself with the capsid for enabling the entry of the DNA into the procapsid, pUL89 is mainly required to effect DNA cleavage. Based on the limited efficacy of the current drugs ganciclovir, cidofovir and foscarnet, new antiviral therapeutics appear to be in demand. Inhibitors targeting pUL56 and/or pUL89 may offer an attractive alternative since mammalian cell DNA replication does not involve cleavage of concatameric DNA. Drugs targeted to terminase-like proteins should therefore be safe and highly selective.

Antiviral Agents↗
PubMed2005

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease.

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.

Blotting, Western↗
PubMed1992

Sequence of a 10.7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases.

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 10.7 kb. The sequence was determined using a double-strand sequencing strategy adapted from the random-clone strategy. The segment contains seven non-overlapping long open reading frames, YKL500, 505, 510, 513, 516, 518 and 520 and one tRNA gene. YKL505 and YKL513 are two already sequenced genes, the BAF1/ABF1 and the APN1 genes, respectively. YKL510 exhibits a strong homology to the RAD2 protein and YKL516 is presumably a protein kinase.

Amino Acid Sequence↗
PubMed1994

The use of random-breakage mapping to locate the genes APN1 and YUH1 in the Saccharomyces genome, and to determine gene order near the left end of chromosome XI.

We have used the previously described technique of random-breakage mapping to locate the two yeast genes APN1 and YUH1. The APN1 locus is located approximately 235 kb from the left telomere of chromosome XI, and shows weak (approximately 53 cM) genetic linkage to ura1. The YUH1 locus is located approximately 140 kb from the right telomere of chromosome X, and genetically maps 3.6 cM distal to cdc11. In addition, we show by random-breakage mapping that TRP3 is located approximately 45 kb from the left telomere of chromosome XI, whereas FAS1 is approximately 110 kb from the same telomere. This supports a gene order on the left distal portion of chromosome XI that agrees with other physical reports but is inverted with respect to Edition 11 of the published genetic map. This report confirms that random-breakage mapping is a rapid and convenient method of locating cloned genes.

Chromosome Mapping↗
PubMed2002

Homing at an extragenic locus mediated by VDE (PI-SceI) in Saccharomyces cerevisiae.

PI-SceI (VDE), a homing endonuclease with protein splicing activity, is a genomic parasite in the VMA1 gene of Saccharomyces cerevisiae. In a heterozygous diploid of the VDE-less VMA1 allele and a VDE-containing VMA1 allele, VDE specifically cleaves its recognition sequence (VRS) in the VDE-less VMA1 allele at meiosis, followed by 'homing', i.e. a conversion to a VDE-containing allele. We found that upon VDE expression, homing of a marker gene at an extragenic locus occurs only when a 45 bp element containing the VRS is inserted at its allelic site, while mutants of VDE with no endonuclease activity lack authentic extragenic homing activity. Thus, both the VRS and VDE are required for homing. Insertion of the VRS in a homozygous diploid significantly lowered the spore germination ability, indicating that a template for gene repair at its allelic locus is essential for efficient homing and survival of yeast cells.

Alleles↗
PubMed1993

Identification of deoxyribonuclease II as an endonuclease involved in apoptosis.

Cell death occurs by apoptosis during programmed deletion of cells and following exposure to cytotoxic agents. Central to the mechanism of apoptosis is internucleosomal DNA digestion by an endogenous endonuclease which is thought to mediate cell death. An axiom of apoptosis is that the endonuclease involved is a Ca2+/Mg(2+)-dependent endonuclease. During purification of endonucleases from Chinese hamster ovary cells, we found little Ca2+/Mg(2+)-dependent endonuclease activity, but large amounts of an endonuclease active below pH 7. This acidic endonuclease was activated in intact cells by reducing intracellular pH values below 7 with a proton ionophore. This activity generated internucleosomal digestion of DNA characteristic of apoptosis. Nuclear extracts contained a cation-independent endonuclease with identical pH-dependent activity. We have compared the acidic endonuclease to bovine deoxyribonuclease II (DNase II) and have found them nearly identical by all tests, including sensitivity to various inhibitors, purification by the same chromatographic steps, and recognition by antibody raised against the bovine enzyme. Addition of either the acidic endonuclease or bovine DNase II to isolated nuclei induced internucleosomal DNA digestion up through pH 6.5. These data demonstrate that DNase II can mediate internucleosomal DNA digestion characteristic of apoptosis following intracellular acidification. Furthermore, these data question the premise that the Ca2+/Mg(2+)-dependent endonuclease is the only endonuclease involved in apoptosis.

Acid Phosphatase↗
PubMed1997

Purification, characterization, and amino acid sequencing of DNase gamma from rat spleen.

An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+. Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer. The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.

Amino Acid Sequence↗
PubMed1997

DNA damage, gadd153 expression, and cytotoxicity in plateau-phase renal proximal tubular epithelial cells treated with a quinol thioether.

2-Bromo-bis-(glutathion-S-yl)hydroquinone [2-Br-bis-(GSyl)HQ] causes DNA single-strand breaks (SSB), causes growth arrest, induces the expression of gadd153 (a gene inducible by growth arrest and DNA damage), and decreases histone H2B mRNA in log-phase renal proximal tubular epithelial cells (LLC-PK1). Renal epithelial cells in vivo normally exhibit a low mitotic index, therefore experiments in both plateau- and log-phase cells are necessary for a comprehensive understanding of the stress response to 2-Br-bis-(GSyl)HQ. In the present article we demonstrate that not all features of the stress response in log-phase cells are reproduced in plateau-phase cells. Thus, although 2-Br-bis-(GSyl)HQ causes concentration and time-dependent increases in DNA SSB, and increases the expression of gadd153, histone H2B mRNA levels are unaltered in plateau-phase cells. The relationship between reactive oxygen species, DNA damage, gene expression, and cytotoxicity was also investigated. Our findings suggest that (i) 2-Br-bis-(GSyl)HQ-mediated DNA damage in LLC-PK1 cells is mediated by the generation of H2O2; (ii) DNA damage, either directly or indirectly, contributes to cell death; and (iii) DNA damage, either directly or indirectly, provides the initial signal for gadd153 expression. In addition, DNA repair is rapid in LLC-PK1 cells, and the DNA-repair inhibitors 1-beta-D-arabinofuranosylcytosine and hydroxyurea have no effect on the amount of DNA SSB. Although the addition of 3-aminobenzamide following 2-Br-bis-(GSyl)HQ exposure has no effect on the removal of DNA SSB, it causes a slight but significant increase in gadd153 expression and cell viability, indicating that activation of poly(ADP-ribose)polymerase may exacerbate toxicity. Finally, aurintricarboxylic acid did not prevent DNA SSB or cytotoxicity in 2-Br-bis-(GSyl) HQ-treated LLC-PK1 cells, implying that activation of endonucleases does not play a role in these processes.

Animals↗
PubMed1995

Characterization of endonucleolytic activity of HIV-1 integrase using a fluorogenic substrate.

Retroviruses require viral DNA to be synthesized by reverse transcription in the cytoplasm followed by integration of the resulting viral DNA into the host chromosome in the nucleus. Reverse transcription and integration, essential steps in the life cycle of retroviruses, are possible targets in the development of antiviral reagents. One attractive target is the integrase protein, a product of the retroviral pol gene which is solely responsible for the retroviral integration process through cutting and joining reactions. When screening for massive numbers of antiviral agents, a rapid and precise assay is ideal. We report the application of fluorescence resonance energy transfer (FRET) with fluorescein and eosin as the energy transfer pair to characterize HIV-IN-mediated DNA cleavage reactions. Past concerns with applications of FRET to DNA were due to interactions of the fluorophore with the DNA, resulting in quenched fluorescence. However, in this study these concerns have been resolved with the use of a nucleotide analog with a 12-carbon linker arm, 5-amino (12)-2'-deoxyuridine beta-cyanoethyl phosphoramidite. Steady-state fluorescence studies show that cleavage of the fluorogenic substrate by integrase results in enhancement of quenched donor fluorescence intensity. The fluorescence assay was confirmed by autoradiographic analysis of the cleavage reaction with radiolabeled fluorogenic substrate. This fluorescence assay will facilitate both detailed kinetic studies and the rapid screening of novel integrase inhibitors.

Base Sequence↗
PubMed1996

Excision of oxidative cytosine modifications from gamma-irradiated DNA by Escherichia coli endonuclease III and human whole-cell extracts.

The possible release from gamma-irradiated DNA of eight oxidatively modified cytosine bases by Escherichia coli endonuclease III was examined by trimethylsilylation and gas chromatography/electron impact/mass spectrometry. The results indicated that endonuclease III induced the release of 5-hydroxyhydantoin (1), 5-hydroxyuracil (2), cis-uracil 5,6-glycol (3), 5-hydroxycytosine (4), trans-uracil 5,6-glycol (5), and trans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (8). The release of these products increased with the initial amount of damage in DNA, i.e., the dose of gamma-radiation (0-100 Gy), giving 4.6 +/- 1.0 fmol of 1, 5.8 +/- 0.3 fmol of 2, 4.9 +/- 0.5 fmol of 3, 11.2 +/- 1.2 fmol of 4, 10.7 +/- 2.1 fmol of 5, and 1.5 +/- 0.5 fmol of 8, per microgram DNA per 10 Gy. In addition, we estimated that the relative rates of excision were 5 approximately equal to 3 > (1.2-fold) 1 > (1.5-fold) 4 > (3.3-fold) 2 on the basis of their initial yields in DNA and initial rates of release as a function of incubation time. The excision of 5-hydroxyuracil (2) and 5-hydroxycytosine (4) lesions was studied in greater detail by enzymatic digestion and HPLC coupled to electrochemical (EC) detection which determines the amounts of these products in DNA. The results showed that the excision of 4 was more efficient than that of 2 (2.7-fold) with greater than 50% of the lesions remaining in DNA after treatment. Finally, we examined the excision of products 2 and 4 from irradiated DNA (50 Gy) by whole human cell extracts. The release of product 2 into the hydrosylate was 5.2 +/- 1.4 fmol per microgram of DNA as measured by fluorobenzylation coupled to gas chromatography/electron capture negative-ion chemical ionization/mass spectrometry. In identical samples, the amount of product 2 was reduced by 45.0 +/- 2.6% (225 from 500 fmol per microgram of DNA) and that of product 4 by 7.0 +/- 3.1% (42 from 600 fmol per microgram of DNA) as measured by HPLC/EC analysis.

Animals↗
PubMed1996

Homogeneous enzyme-based binding assay for studying glycosaminoglycan interactions with macromolecules and peptides.

A simple and rapid homogeneous enzyme-based binding assay is described to study the degree of interaction between glycosaminoglycans and various macromolecules/peptides. The method is based on the homogeneous inhibition of a highly positively charged enzyme, acid deoxyribonuclease II (EC 3.1.22.1), by glycosaminoglycan polyanions, such as heparin, chondroitin 4-sulfate, and dermatan sulfate. Catalytic activity of DNase II is inhibited to nearly 100% by relatively small amounts of these glycosaminoglycan molecules. In the presence of species that bind these polyanions, the activity of the enzyme is regained in an amount proportional to the concentration of the species present. Thus, the relative binding affinities of various species with a given GAG can be assessed rapidly by comparing the concentration of the compound required to reverse the enzyme inhibition to 50% of the maximum value (ED(50) values). The feasibility of this binding assay principle is demonstrated by measuring the ED(50) values of five macromolecules: polylysine, polyarginine, protamine, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), using heparins of different size, as well as chondroitin 4-sulfate and dermatan sulfate as the GAG polyanions. The applicability of the assay method is further extended to study GAG-peptide interactions. A variety of small synthetic peptides (8-13 amino acid residues) derived from the heparin-binding domains of protamine and type IV collagen are used as model peptide species. Relative GAG-binding affinities of these macromolecules/peptides are compared to previous literature values and data obtained via a new electrode-based titration method.

Binding, Competitive↗
PubMed1996

A quantitative microtiter plate nuclease assay based on ethidium/DNA fluorescence.

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellular Serratia marcescens endonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.

Animals↗
PubMed1997

Assay method for femtogram order of Ca2+, Mg(2+)-dependent endonuclease.

A sensitive method for the assay of Ca2+, Mg(2+)-dependent endonuclease was developed. The assay procedure is composed of two parts: (i) microscale endonuclease digestion of highly polymerized calf thymus DNA and (ii) the quantification of DNA breaks by measuring the activation of poly(ADP-ribose) polymerase, which is known to be activated proportionally to the number of nicks and ends of DNA added in the reaction mixture. This method was approximately 10(5)-fold more sensitive than a conventional DNase assay detecting acid-soluble DNA formation and, thus, the activity of 20 to 100 fg of purified bull seminal Ca2+, Mg(2+)-dependent endonuclease could be reliably measured. Ca2+ and Mg2+ requirements and the response to histone H2B of the endonuclease were also demonstrated by this method. Using this method, the assay of a very small amounts of Ca2+, Mg(2+)-dependent endonuclease in crude extracts of calf thymus chromatin was possible. This method may be applied to other types of endonucleases by modifying the mixture for endonuclease reaction.

Animals↗
PubMed1993

Properties of an xeroderma pigmentosum revertant cell line expressing endonuclease V.

We have developed a set of cell lines to help distinguish the sequelae of specific lesions in DNA after UV irradiation. Irradiation results in two primary lesions: cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The contributions of each to mutation are considered utilizing a spectrum of cell lines with increasing abilities to repair these lesions. In particular, we focus on a revertant of the XP12Ro(M1) cell line from a patient with Xeroderma pigmentosum, XP129, which is capable of repairing (6-4) photoproducts but not cyclobutane dimers. We have successfully introduced the denV gene into these cells which confers the ability to repair cyclobutane dimers. By comparing the results of a shuttle vector mutation experiment with the vector pZ189, we can correlate specific mutations to specific lesions.

Base Sequence↗
PubMed1993

Cadmium and zinc mediated changes of the Ca(2+)-dependent endonuclease in apoptosis.

Isolated nuclei from mammalian cells contain a Ca(2+)-dependent endonuclease [1]. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis [2]. We report here that zinc inhibits the DNA fragmentation in dependence of the free Ca2+ concentrations, suggesting that a balance between zinc and calcium might regulate the Ca(2+)-dependent endonuclease. Incubation of nuclei with different free calcium concentrations combined with cadmium shows a stronger inhibition of the DNA fragmentation than zinc. Cadmium inhibits the endonuclease in a calcium-independent way. Surprisingly cadmium alone is able to stimulate the endonuclease, thus to replace Ca2+.

Animals↗