MMS-induced mutagenesis and DNA repair in Escherichia coli dnaQ49: Contribution of UmuD′ to DNA repair

dnaQ-encoded epsilon subunit of DNA polymerase III, possesses 3′,5′ exonuclease (proofreading) activity, and is a fidelity factor of polymerase III holoenzyme. It is assumed that during SOS-induced mutagenesis, UmuD′, UmuC and RecA may suppress DnaQ proofreading activity, and allow for translesional DNA synthesis at the cost of fidelity of replication. In this report SOS-dependent, MMS-induced mutagenesis and DNA repair were tested in E. coli dnaQ49 strains. Bacteria were transformed with various pDNAs harboring compilation of the umuD(D′)C genes, and the influence of plasmids on mutagenesis (argE3 → Arg⁺) and DNA repair was tested. DNA damage and repair were tested in plasmid DNA grown in MMS-treated bacteria and isolated either immediately after MMS treatment, or after starving the cells (MFD conditions) for 30 and 60 min, then nicking activity of Fpg protein on plasmid DNAs was analyzed. It has been found that (i) repair of MMS-induced lesions depends on umuD′C, umuD′ (and to much less degree, on umuDC) genes encoded in pDNA; (ii) MMS-induced mutations, in contrast to DNA repair, are highest in the cells transformed with pDNA harboring umuDC, and lowest or zero in cells with plasmids harboring umuD′C. It is postulated that UmuD′C or UmuD′ proteins play a role in the repair of damaged DNA and/or in maintenance of DNA integrity. The kinetics of these processes (perhaps due to introducing too many of the lessions) seems to be different in E. coli dnaQ⁺ and dnaQ cells, and probably this is a reason that (iii) MMS-induced mutations in dnaQ49 strains are not subject to MFD.     

Detection and quantitation of gallid herpesvirus 1 in avian samples by 5′ Taq nuclease assay utilizing Minor Groove Binder technology

A 5′ Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0×10^?2 median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5′ Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.     

Sensitivity of brome mosaic virus RNA1 replication to mutations in the 3′ tRNA-like structure implies a requirement for sustained synthesis of replicase protein 1a

Summary. The replication competence of a series of brome mosaic virus (BMV) RNA1 variants with defined mutations in the 3′ tRNA-like structure, previously characterized in vitro to be defective in minus-strand synthesis and several tRNA-associated functions, was analyzed in barley protoplasts. Inocula containing wild type RNAs2 and 3 and RNA1 bearing either Δknob or 5′Psk mutation failed to replicate. Two additional RNA1 variants, each bearing either M4 or 5′AGA mutation, resulted in detectable accumulation of progeny but are inhibitory to overall viral replication when supplied in high concentrations. Another aminoacylation-defective mutation Δ5′ supported viral replication but did not interfere with viral replication even at higher concentrations. Coinoculation of replication-incompetent variants of RNAl with wt RNAs2 and 3 to Chenopodium hybridum plants resulted in the delayed development of local necrotic lesions characteristic of a wt infection. Sequence analysis of progeny RNA recovered from these lesions indicated that, in each case, a functional 3′ noncoding sequence was restored due to homologous recombination with a corresponding sequence from wt RNA3. Taken together the results suggest that, unlike protein 2a which is required in catalytic amounts, the intrinsic involvement of protein 1a at various stages of virus infection cycle demands its sustained synthesis.     

Overexpression of N-methylpurine-DNA glycosylase in Chinese hamster ovary cells renders them more sensitive to the production of chromosomal aberrations by methylating agents — a case of imbalanced DNA repair

The main N-alkylation products induced in DNA by methylating mutagens (7-methylguanine, 3-methyladenine, 3-methylguanine) are removed by excision repair involving, in the first step of the repair pathway, N-methylpurine-DNA glycosylase (MPG). To elucidate the significance of excision repair of N-alkylpurines in the defense of cells against alkylating agents we have modulated the efficiency of removal of N-methylpurines in Chinese hamster cells by transfecting them with the human MPG cDNA cloned into a mammalian expression vector. Although the stably transfected cells had a significantly higher capacity for removal of N-methylpurines from DNA, they did not gain protection against the cytotoxic and mutagenic effect of alkylating agents. The cells even responded more sensitively with respect to SCE formation. Here we show that the frequency of chromosomal aberrations induced by methyl methanesulfonate and N-methyl-N′-nitro-N-nitrosoguanidine is significantly enhanced in the transfectants. Furthermore the transfectants showed a stronger inhibition of DNA replication and a higher yield of DNA breaks, as measured several hours after methylating agent exposure. The data suggest that overexpression of MPG causes an imbalance in the multi-step process of excision of N-methylpurines from DNA giving rise to a high yield of apurinic sites and/or gapped DNA that are intermediates in the formation of chromosomal aberrations, SCEs and the inhibition of replication in cells exposed to alkylating agents.     

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although ∼600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 5′ termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 5′-to-3′ exonuclease Xrn1, which has specificity for RNAs with 5′ monophosphate.     

Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase α: evidence for a multifunctional role of Mcl1 in DNA replication and repair

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2 and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1⁺, which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase . Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.     

Chromatin assembly factor‐1 (CAF‐1)‐mediated regulation of cell proliferation and DNA repair: a link with the biological behaviour of squamous cell carcinoma of the tongue?

AIMS: Squamous cell carcinoma (SCC) of the tongue shows aggressive behaviour and a poor prognosis. Clinicopathological parameters fail to provide reliable prognostic information, so the search continues for new molecular markers for this tumour. Chromatin assembly factor-1 (CAF-1) plays a major role in chromatin assembly during cell replication and DNA repair and has been proposed as a new proliferation marker. The aim of this study was to investigate its expression in SCC of the tongue. METHODS AND RESULTS: The immunohistochemical expression of the p60 and p150 subunits of CAF-1 were evaluated in a series of SCCs of the tongue. The findings were correlated with the expression of proliferation cell nuclear antigen (PCNA) and patients' clinicopathological and follow-up data. CAF-1/p60 was expressed in all the tumours, whereas CAF-1/p150 was down-regulated in a number of cases. Overexpression of CAF-1/p60 and down-regulation of CAF-1/p150 identified SCCs with poor outcome, in addition to the classical prognostic parameters. CONCLUSIONS: Simultaneous CAF-1-mediated deregulation of cell proliferation and DNA repair takes place in aggressive SCC of the tongue. Therefore, the evaluation of CAF-1 expression may be a valuable tool for evaluation of the biological behaviour of these tumours. This may be relevant to the introduction of improved follow-up protocols and/or alternative therapeutic regimens.     

Generalized resistance to thyroid hormone: Identification of a novel c-erbAβ thyroid hormone receptor variant (leu450) in a Japanese family and analysis of its secondary structure by the Chou and Fasman method

Summary Generalized resistance to thyroid hormone (GRTH) is characterized by elevated circulating levels of thyroid hormone in the presence of a eumetabolic state and failure to respond to triiodothyronine. Various point mutations in the c-erbA thyroid hormone receptor gene are known to be responsible for different phenotypes of GRTH. We herein report a new c-erbA variant in a Japanese family. The variant consisting of a cytosine to adenine base substitution at nucleotide position 1650 altered phenylalanine to leucine in codon 450 in the T₃-binding domain of c-erbA. This base substitution was found in one allele of the 2 affected members of the family. Thein vitro translation products of this mutant c-erbA gene demonstrated a significantly reduced T₃-binding affinity. The secondary structure of this mutant thyroid hormone receptor predicted by the Chou and Fasman method included a new turn in the helix structure in the T₃-binding domain. We also discuss the secondary structures of the previously reported mutant receptors.This paper was previously presented in part at the 1st International Union of Biochemistry and Molecular Biology Conference, 1–6 June, 1992, in Nagoya, Japan.     

Induction of apoptosis by A3 adenosine receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide in human leukaemia cells: a possible involvement of intracellular mechanism

Aim: The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N⁶-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) was studied. Methods: To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. Results: We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp. Conclusion: Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR.     

Dopamine, norepinephrine, and the management of sensorimotor bindings: individual differences in updating of stimulus–response episodes are predicted by DAT1, but not DBH5′-ins/del

Evidence suggests that the flexibility of managing (creating and updating) stimulus–response bindings is driven by the dopaminergic system. Given that striatal dopamine (DA) plays a crucial role in the updating of working memory, the present study tested whether individual differences in the efficiency of updating stimulus–response episodes (event files) are predicted by differences in genetic predisposition related to the efficiency of the striatal dopaminergic pathway. In view of contrasting claims that stimulus–response binding is related to norepinephrine, we also considered genetic predispositions regarding noradrenergic pathways. In a sample of 100 healthy adults, we studied whether the degree to which stimulus–response bindings affect ongoing performance is predicted by polymorphisms of the dopamine transporter gene (DAT1, associated with striatal DA levels) and DBH5′-ins/del (strongly correlated with dopamine beta-hydroxylase, the enzyme catalyzing the dopamine-norepinephrine conversion). The performance of 9-repeat carriers of the DAT1 gene was more affected by stimulus–response bindings than the performance of 10/10 homozygotes was, while DBH5′-ins/del polymorphism was not related to performance. This outcome pattern suggests a crucial role of the nigrostriatal dopaminergic pathway in the flexible management of stimulus–response episodes, whereas norepinephrine does not seem to play a role.     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: cytochemical localization of a NTPDase1 and an ecto-5′-nucleotidase and effects of adenine nucleotides on cellular viability

Nucleoside triphosphate diphosphohydrolase 1 (NTPDase1), which hydrolyzes extracellular ATP and ADP, and ecto-5-nucleotidase, which hydrolyzes AMP, are characterized for Trichomonas vaginalis. Ultrastructural cytochemical microscopy showed NTPDase1 and ecto-5-nucleotidase activities on the surface of the parasites. High levels of extracellular adenine nucleotides and adenosine did not exert cytolytic effects in intact cells of T. vaginalis. Our results suggest that these enzymes are relevant for the survival of the parasite during exposure to extracellular nucleotides. Since the ecto-localization of these enzymes is essential for the maintenance of adenosine extracellular levels, this nucleoside could be important for the purine salvage pathway in the parasite.     

Suppression of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">rad27</Emphasis> null mutant phenotypes by the 5′ nuclease domain of <Emphasis Type="Italic">Escherichia coli</Emphasis> DNA polymerase I

RNA primer removal from Okazaki fragments during lagging-strand replication and the excision of damaged DNA bases requires the action of structure-specific nucleases, such as the mammalian flap endonuclease 1 (FEN-1). This nuclease contains two conserved motifs enriched with acidic amino acid residues that are important for catalytic function. Similar motifs have been identified in nucleases found in viruses, archebacteria, eubacteria, and in eukaryotes ranging from yeast to humans. Unique among these proteins, the putative FEN-1 homologue in Escherichia coli is contained within the N-terminal region of the DNA polymerase I (PolN). To demonstrate that the cellular functions of FEN-1 reside in PolN, we cloned and expressed the amino terminal domain (323 amino acid residues) of PolI in a Saccharomyces cerevisiae strain lacking the FEN-1 homologue RAD27. Overexpression of PolN suppressed, to varying degrees, phenotypes associated with a rad27 null strain. These include temperature sensitivity, Okazaki fragment processing, a mutator phenotype, a G2/M cell cycle arrest, minichromosome loss, and methyl methane sulfonate sensitivity. We purified Rad27 and PolN proteins in order to determine whether differences in their intrinsic nuclease activities or interaction with proliferating cell nuclear antigen (PCNA) could explain the partial suppression of some phenotypes. We found that the in vitro nuclease activities of Rad27 were more potent than those of PolN and the activity of Rad27, but not PolN, was stimulated by PCNA. We conclude that the N-terminal nuclease domain of E. coli polymerase I encodes a functional homologue of FEN-1.     

Absence of modulation of monokine production via endogenous cyclooxygenase or 5-lipoxygenase metabolites: MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), indomethacin,or arachidonate fail to alter immunoreactive interleukin-1β, or TNF-α production by human monocytes in vitro

Human peripheral blood monocytes exposed to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid) at doses which abolish formation of 5-lipoxygenase metabolites showed unaltered interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α) levels in response to phorbol ester, concanavalin A, serum-treated zymosan, or lipopolysaccharide. Indomethacin (10 μM), alone or in combination with MK-886, also failed to modulate monokine production in response to any stimulus. Exogenous arachidonate (3–30 μM) which augmented the formation of PGE₂ and LTB₄ in the absence of stimulation, also had no effect on monokine production. LPS-induced IL-1 and TNF production occurred despite stimulation of PGE₂ synthesis. The results make a role for endogenous prostaglandins and leukotrienes in the regulation of monocyte IL-1β and TNF-α production unlikely. These data also indicate that MK-886, a novel inhibitor of 5-lipoxygenase product formation, is a potentially useful leukotriene inhibitor which does not affect monokine production.     

Erratum to: To the article “Preventive Efficacy of Oxidized Dextran and Pathomorphological Processes in Mouse Lungs in Avian Influenza A/H5N1” by O. V. Potapova, V. A. Shkurupiy, T. V. Sharkova, A. V. Troitskiy, N. G. Lusgina, and A. M. Shestopalov, Vol. 150, No. 6, pp. 707-710, April, 2010

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