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PubMed1978

Ribonuclease H from rat liver. II. Partial purification and characterization of cytosol ribonuclease H1.

We have detected in rat liver cytosol three enzymes (termed C-1, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-1 and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-1, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg2+ and Mn2+. Cleavage by C-1 and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-1 is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.

Animals↗

PubMed2026

Quercetin, a flavonoid, suppresses viral proliferation by interfering with the ubiquitin transfer from E1 to E2 enzymes.

Quercetin is recognized for diverse pharmacological activities. However, the mechanism underlying its broad-antiviral effects has not been elucidated. Herein, we identified quercetin as a potent inhibitor of both double-stranded DNA virus Bombyx mori nucleopolyhedrovirus (BmNPV) and single-stranded RNA virus porcine reproductive and respiratory syndrome virus (PRRSV). Surface plasmon resonance (SPR) revealed that quercetin targets host ubiquitin-activating enzyme 1 (Uba1) homologs. Uba1 knockdown reduced viral proliferation and enhanced the antiviral effect of quercetin, whereas Uba1 overexpression functioned oppositely. Quercetin bound Uba1 homologs with high affinity. Notably, mutation of two binding residues, Q977 and G978, significantly disrupted the binding between BmUba1 and quercetin, and abolished quercetin's antiviral activity. Quercetin obstructed the transfer of ubiquitin from Uba1 to the E2 enzyme Ubc6, impairing the ubiquitination process. Similarly, quercetin inhibited PRRSV proliferation via targeting Uba1 in mammals. These findings elucidate the molecular mechanism underlying the pharmacological effects of quercetin, providing a theoretical basis for the development of novel antiviral agents against both DNA and RNA viruses.

Quercetin↗

PubMed2025

Targeting RAD52 overcomes PARP inhibitor resistance in preclinical Brca2-deficient ovarian cancer model.

BRCA-mutated ovarian cancer commonly develops resistance to poly (ADP-ribose) polymerase (PARP) inhibitors. Here, we investigated the DNA repair protein RAD52 as a potential target to overcome resistance. In analysis of The Cancer Genome Atlas datasets and immunohistochemistry of tissue microarrays, elevated RAD52 expression correlated with poor overall survival in patients with high-grade serous ovarian cancers. We tested two PARP inhibitor-resistant Brca2-deficient mouse ovarian cancer models, ID8-OR and HGS2-OR. HGS2-OR cells had higher RAD52 expression than parental lines. Rad52 knockout or knockdown restored PARP inhibitor sensitivity in both models. In syngeneic mice, ID8-OR cells in which Rad52 was knocked out yielded lower tumor burden and longer overall survival than control cells. Rad52 depletion impaired single-strand annealing and homologous recombination and led to accumulation of DNA double-strand breaks after PARP inhibitor treatment. RNA sequencing demonstrated that PARP inhibitor treatment induced Polq expression in Brca2- and Rad52-deficient cells, suggesting a switch to microhomology-mediated end joining. Finally, the RAD52 inhibitor D-I03 synergized with a PARP inhibitor to reduce cell viability and tumor burden and prolong survival. Collectively, our findings establish RAD52 as a promising therapeutic target to overcome PARP inhibitor resistance in BRCA2-mutated ovarian cancer and offer mechanistic insights to inform future clinical strategies.

Journal Article↗

PubMed1976

Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.

Bacteria↗

PubMed2026

Recurrent patterns of TOP1-mediated neuronal genomic damage shared by major neurodegenerative disorders.

Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) represent two major categories of neurodegenerative disorders-TAR DNA-binding protein 43 (TDP-43) and tau proteinopathies-for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 469 neurons from C9ORF72 ALS, C9ORF72 FTD, AD, and control brains revealed increased somatic single-nucleotide variants (sSNVs) and insertions/deletions (sIndels) in all three diseases. Mutational signature analysis identified a disease-associated sSNV signature consistent with oxidative damage and an sIndel process affecting 22% of ALS, 76% of FTD, and 61% of AD neurons-but only 2% of control neurons-resembling signature ID4, previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Rapid approach to DNA adduct recovery (RADAR) assays confirmed increased TOP1-DNA covalent complexes, and duplex sequencing confirmed the increased sIndels and identified single-strand events as likely precursor lesions. TOP1-associated sIndel mutagenesis and genome instability thus represent a mechanism shared by both TDP-43 and tau neurodegeneration.

Humans↗

PubMed1976

Mung bean nuclease I. Terminally directed hydrolysis of native DNA.

Under conditions which favor the duplex structure of DNA, mung bean nuclease catalyzes a limited number of double-strand cleavages (probably less than 50) in the interior of native T7 DNA. However, under conditions which are not as favorable to a tight helical structure, the large duplex polymers previously produced are completely degraded from their termini with a continuous accumulation of mono-, di-, and trinucleotides. The terminally directed activity is an intrinsic property of the enzyme molecule because (1) it is inactivated and reactivated in parallel with the single-strand activity and (2) the two activities coelectrophorese on analytical gels. Kinetic measurements indicate that the apparent Km for the terminally directed hydrolysis of native DNA is relatively high. The pH optimum for both the hydrolysis of denatured DNA and the terminally directed hydrolysis of native DNA becomes more acidic with increasing salt concentration. The relative preference for single-stranded structures increases as the pH becomes more basic.

DNA↗

PubMed1976

Biochemical characteristics of rat C-type virus WF-1.

The non-oncogenic rat C-type virus WF-1, isolated from a Wistar-Furth rat embryo cell line, was characterized biochemically. The purified virus has a buoyant density of 1.15 to 1.16 g/cm3 in sucrose, RNA-dependent DNA polymerase (reverse transcriptase) activity, and RNA with a sedimentation coefficient of 62 to 68 S. The viral RNA is single-stranded, and, upon treatment with heat, yields components with sedimentation coefficients of 36 S, 18 to 20 S, and 4 to 12 S.

Animals↗

PubMed1975

Replication of IPN virus: a cytochemical and biochemical study in SWT cells.

Although IPN virus failed to multiply at 30 degrees, it replicated at 16 degrees and 22 degrees in SWT cells. At 22 degrees the viral eclipse period lasted nearly 6 hr with maximal virion titers attained by 24 hr, whereas replication at 16 degrees was much slower. The replication of the virion was inhibited by 0.05 mug/ml of AD which did not interfere with the production of reovirus. Biochemical studies revealed that cellular DNA synthesis was markedly reduced (greater than 50%) soon after infection whereas total RNA synthesis was enhanced. The period of rapid increase in RNA synthesis paralleled the exponential production of infectious virus. Viral inclusion bodies, revealed by acridine orange-staining of virus-infected cells (SWT and RGG-2) late in the infectious cycle, were found to contain single-stranded RNA on the basis of their staining characteristics and sensitivity to RNase.

Acridines↗

PubMed1977

Characterization of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells.

The properties of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells were determined. The enzyme has a molecular weight of approximately 40000. Its pH optimum is at pH 4.0, it is not inhibited by ethylenediaminetetraacetate (EDTA), and it is active at temperatures up to 60 degree C. The enzyme cleaves adenovirus DNA in a stepwise manner. The limit digestion product has a molecular weight of 120000-200000. There is evidence that the cleavage reaction proceeds via an initial single-strand nick. Under the conditions tested the endonuclease did not seem to reveal a high degree of specificity as to the recognition of cleavage sites, or else the sites recognized occurred very frequently.

Adenoviruses, Human↗

PubMed1977

Transcriptase activity associated with rabies virion.

Rabies virion-associated transcriptase activity was investigated in vitro and compared with that of the New Jersey serotype of vesicular stomatitis virus. The concentration of detergent that affected [3H]GMP incoporation into acid-insoluble material was significantly different for both viruses. Vesicular stomatitis virus New Jersey required 0.05 to 0.1% nonionic detergent, whereas rabies virion could not be fully activated unless 4 to 5% detergent was used. Other optimal conditions were as follows: 40 mM NaCl, 5 mM Mg2+, 40 mM Tris-hydrochloride (pH 7.4), 5 mM dithiothreitol, and 30 degrees C. The reaction required four nucleoside triphosphates. The initial rate of RNA synthesis by rabies virion enzyme was 140 pmol of GMP incorporated/mg of viral protein per h and linearly increased until about 8 h, with a slight initial lag phase. The enzyme activity that correlated with the content of L protein was highest when rabies virions were grown at 33 degrees C. The product was single-stranded RNA, which was complementary in base sequences to rabies viral RNA. Most of the RNA synthesized sedimented at 6-16S.

DNA-Directed RNA Polymerases↗

PubMed1976

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.

Centrifugation, Density Gradient↗

PubMed1977

[Bacterial recombination].

The most perspective trends in studying the mechanisms of genetic exchanges in bacteria are discussed. They are the following. 1. Comparison of the recombination stages in various biological phenomena such as transformation, transduction (generalized), conjugation (common and single-stranded) and transfection. 2. Characterization of genetic determinants and basic enzymes of the recombination process in Rec-systems of different bacteria. 3. Determination of some peculiar features of the intragenic recombination and finding a possible approach to their research. 4. Analysis of some plastic properties of the DNA structure and their application to the modern ideas in the formulation of a molecular mechanism of genetic recombination.

Bacillus subtilis↗

PubMed1979

Terminal transferase-like activity in human melanoma cells.

An enzyme activity, capable of attaching deoxynucleotides to single-stranded oligonucleotide primers, has been detected in extracts from cultured melanoma cells. The substrate preferences of this activity show it to be distinct from the calf thymus terminal transferase (EC 2.7.7.31).

Animals↗

PubMed2024

Strategic targeting of Cas9 nickase induces large segmental duplications.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

Saccharomyces cerevisiae↗

PubMed1977

Base specificity of polyamine binding to synthetic polynucleotides.

The binding of polyamines and magnesium to synthetic polynucleotides has been studied by gel filtration on a Sephadex G-50 column. Among the single-stranded polynucleotides examined [poly(A), poly(C), and poly(U)], polyamines were found to bind to poly(C) and poly(U) preferentially, while the binding of Mg2+ was greatest with poly(A). Spermine bound to poly(U) was displaced completely by NH4+ but incompletely by Mg2+, while Mg2+ bound to poly(A) was displaced completely be spermine but incompletely by NH4+. The optimal pH for the binding of spermine to poly(U) was found to be about 7.9, while Mg2+ could bind to poly(A) over a broad pH range (7.1--8.7).

Chemical Phenomena↗

PubMed2027

Production of Viral Particles from a Chikungunya Virus Infectious Clone.

Chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus, which poses challenges for its study and genetic manipulation. Because direct mutagenesis of viral RNA genomes is technically impractical, reverse genetics systems are essential tools for investigating viral biology. To enable such approaches, infectious clones containing a full-length cDNA copy of the viral genome are constructed. The cDNA is positioned under the control of a bacteriophage RNA polymerase promoter, allowing commercial RNA polymerases to use the linearized plasmid as a template for the in vitro transcription of full-length viral genomic RNA (gRNA). Importantly, positive-sense viral genomes serve as mRNAs for the translation of viral proteins in a cellular environment, meaning that these transcripts contain all the information required to initiate viral replication. Following transfection into permissive cultured cells, viral proteins are expressed, enabling genome replication and, ultimately, the recovery of infectious particles from the cell supernatant. Here, we describe a detailed procedure for generating CHIKV particles through plasmid linearization, in vitro transcription, and subsequent RNA transfection.

Chikungunya virus↗

PubMed1976

A compact form of double-stranded RNA in solutions containing poly(ethyleneglycol).

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA (dsRNA) adopt a compact form in solutions, containing sufficiently high concentrations of salt (NaCl) and polymer (PEG). However, only in the cases of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of DNA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides.

Circular Dichroism↗

PubMed1979

Properties of Bacillus subtilis ATP-dependent deoxyribonuclease.

A purification procedure described previously resulting in electrophoretically pure Bacillus subtilis ATP-dependent DNAse has now been modified by adding a fractionation stage with Polymin P to permit large-scale isolation of the enzyme. It has been found that the enzyme molecule (Mr = 300000) consists of two large subunits with Mr 155000 and 140000. The purified enzyme has three activities: (1) DNAse on linear single-stranded and double-stranded DNAs (2) DNA-unwinding and (3) ATPase. Circular DNAs were not affected by the enzyme. Study of the dependence of these activities on temperature, pH, and ATP and Mg2+ concentrations has revealed two different states of the enzyme. At low ATP concentrations and alkaline pH, it showed chiefly nuclease action, degrading considerable amounts of DNA to small fragments five residues long on average. At higher ATP concentrations and neutral pH (more physiological conditions) it predominantly unwound DNA. Simultaneously it cut preferentially one of the duplex strands to fragments more than 1000 residues in length. The results obtained suggest that the energy of the enzyme-cleaved ATP is mainly expended on unwinding rather than on degrading DNA molecules.

Adenosine Triphosphate↗