Uptake of isolated plant chromosomes by plant protoplasts

For mass isolation of plant metaphase chromosomes, cultured cells of wheat (Triticum monococcum) and parsley (Petroselinum hortense) were synchronized by hydroxyurea and colchicine treatment. This synchronization procedure resulted in high mitotic synchrony, especially in suspension cultures of parsley in which 80% of the cells were found to be at the metaphase stage. Mitotic protoplasts isolated from these synchronized cell cultures served as a source for isolation of chromosomes. The described isolation and purification method yielded relatively pure chromosome suspension. The uptake of the isolated plant chromosomes into recipient wheat, parsley, and maize protoplasts was induced by polyethylene-glycol treatment. Cytological studies provided evidences for uptake of plant chromosomes into plant protoplasts.Abbreviations PEG polyethylene glycol - HU hydroxyruea - C colchicine - HUC hydroxyurea and colchicine - CIM chromosome isolation medium - TCM Tris chromosome medium     

Hydroxyurea synchronization of bovine fetal spleen cells

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine     

The Coordination of Centromere Replication,Spindle Formation,and Kinetochore–Microtubule Interaction in Budding Yeast

The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome–microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore–microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU), a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore–microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome–microtubule interaction.     

Plant tyrosine decarboxylase can be strongly inhibited by l-α-aminooxy-β-phenylpropionate

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1–1 g · ml^-1 tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.Abbreviations TCA trichloroacetic acid - TM tunicamycin     

Caffeine inactivation of mammalian cells in the S phase of the mitotic cycle due to anomalous replicon initiation during the inhibition of the elongation of replicating DNA

Chinese hamster cells were treated with an inhibitor of DNA synthesis (hydroxyurea or arabinoside-cytesine) in non-toxic concentrations for 20 hours in the presence or absence of caffeine (2 mM). Under these conditions caffeine considerably inactivates the cells. If cells are synchronized by hydroxyurea (0.25 mM) in the S-phase of mitotic cycle, the addition of caffeine kills all the S-phase cells, while gamma-irradiation or novobiocine treatment markedly decreases the sensibilizing effect of caffeine. These findings permit us to conclude that cell inactivation is due to anomalous reinitiation of DNA synthesis stimulated by caffeine in the presence of drugs which inhibit the DNA chain elongation.     

Lack of effect of hydroxyurea on base excision repair in mammalian cells

The effect of hydroxyurea on the initial steps of base excision repair has been examined in mammalian cells in 3 different proliferative states: i.e., quiescent cells, asynchronously growing cells undergoing multiple divisions prior to confluence; and synchronous cell populations undergoing the first cell cycle(s) after release from quiescence. Two parameters of the base excision repair pathway were examined: (1) The direct excision of 7-methylguanine from cellular DNA in the presence of increasing hydroxyurea concentrations was quantitated by high performance liquid chromatography; (2) the effects of hydroxyurea on the uracil DNA glycosylase were examined by quantitating the levels of this base excision repair enzyme in quiescent and proliferating cells. In quiescent cells, hydroxyurea at concentrations routinely used to quantitate DNA repair had no effect on the excision rates of 7-methylguanine examined over a span of 3 days; nor was there any effect on the specific activity of uracil DNA glycosylase in confluent cells. In asynchronously proliferating mammalian cells, identical hydroxyurea concentrations had no effect on the induction of the glycosylase. In synchronous growing cells HU had no effect on the temporal sequence of induction of uracil DNA glycosylase prior to DNA replication, nor on the extent of this induction. These results suggest that hydroxyurea at concentrations generally used to measure DNA repair has no effect on base excision repair.     

Secondary activities of diverse inhibitors potentiate the response of hamster embryo cultures to a mitotic stimulus

The reversal of the density dependent inhibition of replication (DDIR) of Syrian hamster embryo cultures by fresh medium containg 30-50% fetal bovine serum was poorly synchronized. There were two waves of DNA synthesis eight to nine hours apart, which by examination of autoradiograms of cultures pulse-labeled with 3H-thymidine, were found to involve 55% and 45% of the cell population. The second wave was found to be due to a subpopulation of epithelioid cells set in the predominantly fibroblastic cell cultures. Pretreatment of the DDIR cultures with hydroxyurea (HU) or arabinosyl cytosine (ara-C), followed by serum stimulation in the absence of the drugs, led to an enhancement of the synchrony. The effect increased with lengthening of the contact with antimetabolites, to a maximum after 20 hours' pre-exposure, and was in part due to the shortening of the GI phase of the epithelioid elements, and in part to increasing the synchrony of the fibroblastic cells. The resulting synchrony involved some 95% of the cells in simultaneous DNA synthesis after a median Gl period of 12 hours. The effect had no relationship to the role of HU and ara-C as specific inhibitors of DNA synthesis since the cultures were mitotically quiescent, and a similar enhanced response could be induced in DDIR cultures by prestimulation exposure lasting only two hours to cycloheximide (cyx), an inhibitor of protein synthesis. Pre-exposure of DDIR cultures to actinomycin D did not potentiate the cell response. A survey of the known secondary inhibitions caused by these three antimetabolites suggests that they all may cause deficiencies in the glycolipid or glycoprotein moieties of the cell surface. These observation provide a use ful, simple means of improving synchrony in these systems and may prove to be a useful probe for investigating the role of the cell surface in regulating cell replication.     

Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide triphosphates

Hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone, pyrazoloimidazole, 3,5-diamino-1,2,4 triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid were examined for their effects on cellular dNTP pools, DNA excision repair, DNA replication and deoxynucleoside uptake in human diploid fibroblasts. All 7 agents were effective inhibitors of the UV excision repair process in noncycling quiescent cells, but not in rapidly dividing log-phase cells. This differential effect clearly demonstrates dependency upon modulation of cellular purine dNTP pool levels at the level of the reductase. Repair synthesis is shown to be less sensitive to all 7 reductase inhibitors than is replicative synthesis. Studies on cellular uptake of labeled DNA precursors in inhibitor-treated cells support the notion that deoxynucleosides cannot channel into the replicative synthesis process whereas they are readily utilized at repairing sites.Abbreviations HU hydroxyurea - dA deoxyadenosine - TSC pyridine-2-carboxaldehyde thiosemicarbazone - IMPY pyrazoloimidazole - THBA 3,4,5-trihydroxy benzohydroxamic acid - DHBA 3,4-dihydroxy benzohydroxamic acid - UDS unscheduled DNA synthesis - dT thymidine - dNTP deoxynucleoside triphosphate     

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells

Ornithine decarboxylase activity increases at least 4–5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G₁ and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G₁-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.     

Hydroxyurea synchoronization of bovine fetal spleen cells.

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine

The S phase kinetics have been evaluated in cells synchronized with either thymidine or hydroxyurea by direct analysis of the proportion of DNA semi-conservatively replicated as a function of time after release from the inhibitor. The proportion of DNA replicated was determined by growing the cells in medium containing 5-bromodeoxyuridine (BUdR) and subsequently measuring the amount of DNA that acquired increased buoyant density in CsCl gradients. The results confirm previous reports that substantial DNA synthesis occurs during TdR treatment. In contrast, HU provided a population of cells very nearly at the G 1-S interphase since 95 % of the DNA replicated synchronously after its removal. It is proposed that by measuring the rate and maximum extent of DNA replication with BUdR during S phase one can evaluate different synchrony methods for use in experiments designed to study aspects of semiconservative DNA replication.     

Modulation of DNA synthesis in aortic smooth muscle cells by dinitrotoluenes

Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced ³H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1–100 µM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1–100 µM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions.Abbreviation DAT diaminotuluene - tDNT technical grade dinitrotoluene - DNT dinitrotoluenes - HU hydroxyurea - IP intraperitoneal - LDH lactate dehydrogenase - MCT oil medium chain triglyceride - NPTC non-protein thiol content - RDS replicative DNA synthesis - SEM standard error of the mean - SMC smooth muscle cells - UDS unscheduled DNA synthesis     

The N-terminal region of the <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> RecQ helicase,Rqh1p,physically interacts with Topoisomerase III and is required for Rqh1p function

The Schizosaccharomyces pombe rqh1⁺ gene encodes a member of the RecQ DNA helicase family. Members of this protein family are essential for the maintenance of genetic integrity. Thus, mutations in the genes encoding the human RecQ homologues Blm, Wrn and RecQ4 cause Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively—diseases which result from genome instability. S. pombe cells that lack a functional rqh1⁺ gene show reduced viability and display defective chromosome segregation, particularly after UV irradiation or S-phase arrest. In this study we used an rqh1⁺ deletion series to show that the N-terminal portion of Rqh1 is essential for Rqh1 function. Moreover, the conserved Helicase and RNaseD C-terminal (HRDC) domain of Rqh1 also plays a role in allowing cells to tolerate exposure to DNA damaging agents and the S-phase inhibitor hydroxyurea (HU). We also demonstrate that Topoisomerase III (Top3) binds to a site within the first 322 N-terminal amino acids of Rqh1 and that this binding correlates with Rqh1 function. Genetic analysis of rqh1^– top3 mutants reveals that, in the presence of functional or partially functional Rqh1 protein, Top3 is required to maintain genome integrity and cell viability.     

Synergistic effects of hydroxyurea and thymidine on the growth inhibition of V79 cells

The effects of hydroxyurea (HU) and thymidine (TdR) on cell cycle progression in V79 Chinese hamster lung cells were examined by flow cytometry. Suppression of the cell cycle progress rate by HU was further enhanced by the combination of a low concentration of TdR and HU as compared to that induced by TdR alone; i.e., these drugs were shown to have a synergistic effect. It was concluded that the presence of TdR was effective in assisting HU-induced suppression of DNA synthesis.