Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Genetic and biochemical evidences reveal novel insights into the mechanism underlying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Sae2-mediated abrogation of DNA replication stress

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5′ end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2^G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stress-related defects in S. cerevisiae.     

Direct Inhibition of <Emphasis Type="Italic">Col</Emphasis> E<Subscript>1</Subscript> Plasmid DNA Replication in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Rifampicin

COLICINOGENIC factor E₁ (Col E₁) is a small bacterial plasmid (4.2×10⁶ daltons) present in colicinogenic strains of Escherichia coli¹ to the extent of about twenty-four copies per cell (Clewell and Helinski, unpublished results), which continues to replicate in the presence of high levels of chloramphenicol, a specific inhibitor of protein synthesis, although the chromosome only completes current rounds of replication and ceases (Clewell and Helinski, unpublished results). The average rate of Col E₁ semiconservative replication in the absence of protein synthesis is, in certain conditions, faster than (as much as eight times) the normal rate of synthesis (Clewell, unpublished results). Replication continues for 10–15 h after the addition of chloramphenicol, resulting in nearly 3,000 copies of Col E¹ DNA per cell. We are taking advantage of this system to study the effects of a number of antibiotics on DNA replication and now report evidence that rifampicin (an active semisynthetic derivative of rifamycin B)², an antibiotic known specifically to inhibit bacterial DNA dependent RNA polymerase^3–6, has a dramatic inhibitory effect on Col E¹ DNA replication.     

Effect of 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenyl)-azouracil on <Emphasis Type="Italic">B. subtilis</Emphasis> DNA Polymerases

DNA replication in Bacillus subtilis^1,2 and other Gram-positive organisms³ is specifically inhibited by 6-(p-hydroxyphenyl)-azouracil (HPUra). The site of action of this compound has not so far been identified, but important progress was made by Brown et al.⁴, who studied the effect of HPUra on DNA synthesis in B. subtilis cells made permeable to externally supplied deoxynucleoside triphosphates by treatment with toluene. In this in vitro system, HPUra had no inhibitory effect when added alone, but in the presence of NADPH or dithiothreitol (DTT) the drug was reduced to a colourless form which specifically inhibited DNA synthesis.     

Chromosome Replication in Toluenized <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cells

BIOCHEMICAL studies of chromosome replication have been hampered by the unavailability of an adequate in vitro system with the basic features of in vivo DNA replication. The criteria for such a system are: (1) semiconservative replication; (2) normal biological activity of newly synthesized DNA; (3) normal advancement of the original replication fork; (4) rate of DNA replication equivalent to in vivo; and (5) expected phenotypic behaviour of temperature-sensitive dna mutants. Systems in Escherichia coli, a membrane-DNA fraction¹, an agar-embedded cell lysate² and toluene-treated cells³ have met two or three of the requirements. Several laboratories have also reported the expected behaviour of ts-dna E. coli mutants in toluenized cells^3–5.     

Disruption of the gene encoding restriction endonuclease <Emphasis Type="Italic">Sua</Emphasis>I and development of a host–vector system for the thermoacidophilic archaeon <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host–vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N⁴-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose–tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10³/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40–50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.     

Inhibition of Bacterial DNA Replication by 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo)-uracil

THE semi-conservative replication of DNA of Gram-positive bacteria is specifically inhibited by 6-(p-hydroxyphenyIazo)-uracil (HPUra; obtained from ICI) in an apparently novel mechanism^1–4. We have attempted to characterize the HPUra-sensitive site in replication using in vitro preparations of drug-sensitive bacteria. In particulate and soluble preparations of sensitive bacteria, however, HPUra at high concentration does not significantly inhibit polymerization of deoxyribonucleotides^2,4. Since these systems may not accurately represent the process of DNA replication as it occurs in vivo, we have examined the effect of HPUra on a more suitable, toluene-treated preparation of Bacillus subtilis described by Matsushita et al.⁵. In this preparation, DNA replication is ATP-dependent, utilizes deoxyribonucleotides to give biologically active DNA, semi-conservatively and sequentially in the proper gene order. HPUra can inhibit DNA replication by this system. We describe here the characteristics of HPUra inhibition and the conditions necessary for it to occur.     

<Emphasis Type="Italic">E. coli</Emphasis> DNA Polymerase I and other Host Functions participate in T4 DNA Replication and Recombination

The recognition of bacterial functions involved in DNA metabolism of bacteriophage T4 might reveal interactions between different enzymes during DNA replication and recombination. To detect such functions we have studied the replication of complete and incomplete T4 chromosomes in various mutant strains of Escherichia coli that are defective in their own DNA metabolism. We found that several E. coli functions can substitute for phage functions in T4 replication and recombination and will discuss here the role of the E. coli pol A gene which codes for DNA polymerase I^1–4 and of the dna B and E genes^3,5.     

Inhibition of a Discrete Bacterial DNA Polymerase by 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo)-uracil and 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenylazo-)-isocytosine

6-(p-HYDROXYPHENYLAZO)-URACIL (HPUra) specifically inhibits the semi-conservative replication of DNA in Gram-positive bacteria^1–3. We have reported that HPUra inhibits ATP-dependent polymerization of deoxyribonucleotides in vitro in toluene-treated B. subtilis⁴. Further studies of the effect of HPUra and its amino analogue, HPIsocytosine (6-(p-hydroxyphenylazo)-2-amino, 4-keto pyrimidine), in toluene-treated B. subtilis have provided considerable information on the mechanism of arylazopyrimidine action. First, HPUra and HPIsocytosine do not inhibit DNA synthesis unless they first are reduced to their colourless, hydrazo forms (refs. 4 and 5 and Mackenzie, Wright and Brown, unpublished results). Second, the inhibitory action of reduced HPUra and that of reduced HPIsocytosine are completely antagonized, respectively, by dGTP and dATP⁵. Third, drug-resistant mutants have been isolated which catalyse drug-resistant DNA synthesis following their permeabilization with toluene. These observations suggest that reduced HPUra and HPIsocytosine inhibit DNA replication by interfering competitively with the enzymatic polymerization of specific purine deoxyribonucleotides. We examined, therefore, cell free preparations of ^{B. subtilis} in an effort to identify a discrete DNA polymerase as the site of drug action. We report here experiments with crude and partially fractionated extracts of DNA polymerase I-deficient mutants which indicate the existence of at least one drug sensitive polymerase. Bazill and Gross⁶ have independently isolated chromatographically discrete HPUra-sensitive polymerases from extracts of B. subtilis.     

Identification and characterization of a complex chromosomal replication origin in<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

In the budding yeast,S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (±300 bp). In contrast, in mammalian cells andDrosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just likeS. cerevisiae origins. Because the fission yeast,Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than doesS. cerevisiae, we thought thatS. pombe replication origins might prove to resemble — and thus be helpful models for — animal cell origins. An attempt to test this possibility using 2D gel techiques resulted in identification of a replication origin near theura4 gene on chromosome III ofS. pombe. The 2D gel patterns produced by thisS. pombe origin indeed resemble the patterns produced by animal cell origins and show that theS. pombe origin cannot be precisely located. The data suggest an initiation zone of 3–5 kbp. Some aspects of the 2D gel patterns detected at theS. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.     

Cell cycle arrest mediated by WEE1 is involved in the unfolded protein response in plants

Activation of the unfolded protein response (UPR) in mammalian cells leads to cell cycle arrest at the G1 phase (Thomas et al., J Biol Chem 288:7606–7617, 2013). However, how UPR signaling affects cell cycle arrest remains largely unknown in plants. Here, we examined UPR and endoreduplication in Col-0, wee1, and ER stress sensing-deficient ire1a&b plants during DNA replication and ER stress conditions. We found that WEE1, an essential negative regulator of the cell cycle, is involved in the maintenance of ER homeostasis during genotoxic stress and the ER stress hypersensitivity of ire1a&b is alleviated by loss-of-function mutation in WEE1. WEE1-mediated cell cycle arrest was required for IRE1–bZIP60 pathway activation during ER stress. In contrast, loss-of-function mutation in WEE1 caused increased expression of UPR-related genes during DNA replication stress. WEE1 and IRE1 were required for endoreduplication during DNA replication stress and ER stress, respectively. Taken together, these findings suggest that cell cycle regulation is associated with UPR activation in different manners during ER stress and DNA replication stress in Arabidopsis.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Independence of Cell Division and DNA Replication in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

IN Escherichia coli the completion of a round of chromosome replication is necessary before cell division can take place^1,2. A normal cell is therefore unable to divide unless it has at least two chromosomes. If DNA synthesis is specifically inhibited, cell division will continue only until each cell contains a single chromosome. Division then ceases but growth continues so that long filamentous cells are formed³. We describe here the consequences of blocking DNA synthesis in Bacillus subtilis. In this case division of the growing cells continues in spite of the inhibition of DNA replication. Eventually, not only are all pre-existing chromosomes segregated into separate cells but large numbers of cells are formed which contain no DNA.     

Polysaccharide-based silver nanoparticles synthesized by <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> DSM 29614 cause DNA fragmentation in <Emphasis Type="Italic">E. coli</Emphasis> cells

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO₃ to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS^aer and the AgNPs-EPS^anaer, were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS^aer, in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS^aer. In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS^aer exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS^aer. The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag⁺ release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.     

<Emphasis Type="Italic">Streptomyces tunisialbus</Emphasis> sp. nov., a novel <Emphasis Type="Italic">Streptomyces</Emphasis> species with antimicrobial activity

A novel actinomycete strain designated S2^T was isolated from Tunisian rhizosphere soil of Lavandula officinalis. This isolate exhibited broad spectrum antibacterial activity against several Gram-positive and Gram-negative bacteria and also antifungal activity against yeast and filamentous fungi. The isolate S2^T presents morphological and chemotaxonomic characteristics typical of the members of the genus Streptomyces. Whole cell hydrolysates of S2^T were found to contain LL-diaminopimelic acid. The major fatty acids were identified as C16:0, anteiso-C15:0 and iso-C16:0 whereas the predominant menaquinones were found to be MK-9(H6) and MK-9(H8). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and three unidentified compounds. The G+C content of the genomic DNA was determined to be 71.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2^T belongs to the genus Streptomyces and is closely related to Streptomyces netropsis DSM 40259^T with 99.86% sequence similarity. Multi-locus sequence analysis (MLSA) based on four house-keeping gene alleles (gyrB, recA, trpB, rpoB) showed that isolate S2^T is closely related to S. netropsis, with an MLSA distance greater than 0.007. The DNA–DNA relatedness between strain S2^T and its near phylogenetic neighbour was 63.6 ± 2.3%, which is lower than the 70% threshold value for delineation of genomic prokaryotic species. This isolate was also distinguished from the type strain S. netropsis DSM 40259^T, using a combination of morphological and physiological features. Based on its phenotypic and molecular properties, strain S2^T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces tunisialbus sp. nov. is proposed. The type strain is S2^T (= JCM 32165^T = DSM 105760^T).