Kinetics and localization of wound-induced DNA biosynthesis in potato tuber

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive “Click-iT EdU Assay” employing incorporation of the thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU), in conjunction with 4′,6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12 h, rapidly reached a maximum at about 18 h and then declined to near zero at 48 h. About 28% of the nuclei were EdU labeled at 18 h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30 h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7–8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.     

Adeno-associated virus rep protein synthesis during productive infection.

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. We studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with [35S]methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.     

The effect of vinyl chloride monomer,chloroethylene oxide and chloracetaldehyde on DNA synthesis in regenerating rat liver

Vinyl chloride monomer used in the manufacture of polyvinyl chloride is a chemical of increasing industrial importance but has recently been incriminated as a carcinogen, producing a mutagenic effect after being metabolized to active metabolites. The initial effect of vinyl chloride monomer and two of its presumed metabolites, chloracetaldehyde and chloroethylene oxide, on DNA synthesis was investigated in vivo in regenerating rat liver. The established control curve for the DNA synthesis rate after partial hepatectomy demonstrated two waves of synthetic activity at 21 and 30 h. Vinyl chloride, injected intravenously immediately on completion of the operation, depressed the first wave of DNA synthesis by 49.6%. The second peak of DNA synthetic activity was similar to that of the control. Chloracetaldehyde and chloroethylene oxide both produced similar effects on the first wave of DNA synthesis after partial hepatectomy, inhibiting the DNA synthesis rate by approx. 50%. After a regenerating period of 27 h, however, they produced very different effects, chloroethylene oxide raising the control DNA synthesis rate at 30 h by 49% while chloracetaldehyde tended to desynchronize the well-defined second peak of the control. The test compounds have been compared to literature reports of the inhibitory effects of various carcinogens on DNA synthesis.     

Effects of hydroxyurea on DNA synthesis in hairless mouse epidermis

The effect of 5 mg hydroxyurea (HU) i.p. on epidermal DNA synthesis in female hairless mice was assessed by measuring labelling indices and specific activity after 3HTdR injection, flow cytometry (FCM) and cell sorting of prelabelled basal cells. HU causes an almost immediate block in DNA synthesis lasting until 2-2.5 h. During this time the fraction of cells in S remains stationary, 1.20 of normal. From 2.5 to 12.5 h DNA synthesis is resumed, but in cells recruited from G1 or G0. The HU-blocked cells do not move out of S until after 12.5 h. Hence, from 2.5 to 12.5 h, the fraction of cells in S increases to 2.5 of normal, which means that entry into S is open, but exit is blocked. From 12.5 h flux through S is high. The blocked cells are now released and the fraction of cells in S falls to 0.7 of normal at 24.5 h. At 36.5 h a probable new wave of DNA synthesis is indicated. The results also show that 3HTdR is available for at least 20 min after i.p. injection. The consequences of these results for the interpretation of the effect of HU pretreatment on methylnitrosourea skin carcinogenesis are discussed.     

DNA polymerase switching on homotrimeric PCNA at the replication fork of the euryarchaea Pyrococcus abyssi

DNA replication in Archaea, as in other organisms, involves large protein complexes called replisomes. In the Euryarchaeota subdomain, only two putative replicases have been identified, and their roles in leading and lagging strand DNA synthesis are still poorly understood. In this study, we focused on the coupling of proliferating cell nuclear antigen (PCNA)-loading mechanisms with DNA polymerase function in the Euryarchaea Pyrococcus abyssi. PCNA spontaneously loaded onto primed DNA, and replication factor C dramatically increased this loading. Surprisingly, the family B DNA polymerase (Pol B) also increased PCNA loading, probably by stabilizing the clamp on primed DNA via an essential motif. In contrast, on an RNA-primed DNA template, the PCNA/Pol B complex was destabilized in the presence of dNTPs, allowing the family D DNA polymerase (Pol D) to perform RNA-primed DNA synthesis. Then, Pol D is displaced by Pol B to perform processive DNA synthesis, at least on the leading strand.     

Fluocinolone acetonide: A potent inhibitor of mouse skin tumor promotion and epidermal DNA synthesis

The relationship between the inhibition of mouse skin tumor promotion and the inhibition of epidermal DNA synthesis by the steroidal anti-inflammatory agent, fluocinolone acetonide (FA), was investigated. Simultaneous doses of either 10, 1, or 0.1 μg of FA and phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in an almost complete inhibition of promotion, whereas 0.01 and 0.001 μg of FA resulted in inhibition rates of 82% and 15%, respectively. Likewise, simultaneous doses of 10 or 1 μg of fluclorolone acetonide (FCA) and TPA caused a nearly complete inhibition of promotion, whereas 0.1 μg of FCA decreased promotion by 62%. In general, as the dose of both steroids was increased, an increase in the tumor latency period was observed. With the exception of the borderline effect of 0.001 μg of FA, the above doses of FA inhibited epidermal DNA synthesis by at least 60% for a 24-h period. Topical treatment with 10 μg of FA resulted in an almost complete inhibition of DNA synthesis for 6 days. The administration of 10 μg of FA 24 h after TPA treatment brought about a maximal inhibition of DNA synthesis of 65%, as compared with a 98% inhibition in control mice whose DNA synthesis had not been prestimulated. That is, FA was not quite as effective on S-phase cells as on G-1 cells. There appears to be a relationship between the inhibition of tumor promotion and epidermal DNA synthesis.     

Some properties of chromatin synthesized by mouse-L-cells temperature-sensitive in DNA replication

When ts A1S9 mouse L-cells are incubated at the nonpermissive temperature (38.5 degrees) DNA synthesis proceeds at the normal rate for 6 to 8 h; it then declines to attain 1 to 5% of this rate after 24 h. General protein synthesis from precursor leucine is relatively unaffected by the high temperature. In contrast, protein formation from lysine (and arginine) remains unchanged for 12 to 15 h after temperature upshift. It then drops and plateaus at about 25% of the initial rate after 32 h. The chromatin protein and DNA are fully conserved in ts A1S9 cells incubated at 38.5 degrees for at least 24 h after full expression of the ts defect. Temperature inactivation of the ts A1S9 gene product results in inhibition of de novo formation of chromatin. This is evidenced by coordinate suppression of incorporation of dThd and of lysine and arginine into chromatin-bound DNA and histone, respectively.     

IGF-I mediates the stimulatory effect of high phosphate concentration on osteoblastic cell proliferation

Although high concentrations of inorganic phosphate (Pi) are known to have a distinct anabolic effect on bone structure and metabolism, the precise mechanism by which phosphate possesses anabolic effect on bone formation has not been elucidated. The present study was performed to examine the effects of an increase in extracellular Pi concentration ([Pi](e)) on the proliferation of osteoblastic MC3T3-E1 cells. Increase in [Pi](e)(2-4 mM) dose-dependently stimulated DNA synthesis. Indomethacin, an inhibitor of prostaglandin synthesis, did not affect high [Pi](e)-induced DNA synthesis. DNA synthesis first increased affer a 3 h exposure to 4 mM [Pi](e) and its stimulatory effect was observed in a time-dependent manner up to 24 h. On the other hand, DNA synthesis was significantly but partially blocked by cycloheximide, suggesting that this stimulatory effect of high [Pi](e) was at least in part dependent on new protein synthesis. There is recent evidence that MG3T3-E1 cells constitutively produce and secrete insulin-like growth factor-I (IGF-I) and possess IGF-I receptors. IGF-I antiserum (1:10,000 to 1:100) significantly but partially blocked the stimulatory effect of [Pi](e) (4 mM) on DNA synthesis in a concentration-dependent manner. A neutralizing IGF-I antibody as well as IGF-I receptor antibody also significantly but partially blocked DNA synthesis stimulated by high [Pi](e) in a concentration-dependent manner, indicating that IGF-I at least in part mediated the high [Pi](e)-induced effect. Actually, high [Pi](e) significantly increased the secretion of immunoreactive IGF-I into the medium as well as the expression of IGF-I mRNA. Present findings indicate that an increase in [Pi](e) stimulated DNA synthesis partly via an increase in IGF-I action.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Molecular genetics of herpes simplex virus. VIII. further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle.

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.     

Inhibition of S-phase progression in macrophages is linked to G1/S-phase suppression of DNA synthesis genes.

Some of the important controlling events regulating eukaryotic S-phase progression are considered to occur late in the G1 stage of the cell cycle. We show here that stimulation of DNA synthesis in bone marrow-derived macrophages (BMM) by macrophage CSF-1 is preceded by G1 expression of three genes which encode proteins associated with the DNA synthesis machinery--the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear Ag (PCNA). Increased expression for these genes correlated well with the mitogenic response and sustained expression required de novo RNA and protein synthesis and also the presence of CSF-1 for at least most of G1. Inhibitors of BMM proliferation (LPS, TNF-alpha, IFN-gamma, and cAMP elevating agents) suppressed CSF-1-induced expression of M1, M2, and PCNA mRNA measured at 22 h. This suppression occurred even when added up to 12 h after the CSF-1, a period coinciding with the G1/S-phase boundary. The delayed kinetics of this effect parallels the ability of these agents to maximally inhibit CSF-1-induced BMM DNA synthesis when added at similar times. Decreased expression of M1, M2, and PCNA was not merely a consequence of DNA synthesis inhibition because the S-phase inhibitor, hydroxyurea, did not suppress CSF-1-induced gene expression. These results suggest that inhibition of DNA synthesis by antiproliferative agents involves inhibition of expression of several genes associated with the DNA synthesis machinery.     

The role of deoxynucleoside triphosphate pools in the inhibition of DNA-excision repair and replication in human cells by hydroxyurea

The effects of hydroxyurea (HU) on the DNA-excision repair process in human cells has been systematically examined. It is demonstrated that HU induces DNA single-strand break accumulation in a dose-dependent fashion in ultraviolet-irradiated and MMS-treated confluent but not log-phase fibroblasts and that these breaks are clearly the consequence of the inhibition by HU of the enzyme, ribonucleotide reductase. The breaks form rapidly, are stable for at least 10 h and largely disappear by 20 h. The production of these DNA-strand breaks is antagonized by a combined treatment of 10 microM deoxyadenosine, deoxycytidine and deoxyguanosine whereas thymidine potentiates strand-break formation at low HU concentrations. It is also confirmed that HU, while inhibiting replicative synthesis has no apparent inhibitory effect on unscheduled DNA synthesis (UDS) although the increased uptake of labeled DNA precursors into HU-treated cells makes it difficult to assess the actual effects on the repair-synthetic process. Analysis of the effects of HU on deoxynucleoside triphosphate pool levels and the demonstration of the failure of the HU block to replicative synthesis to be reversed by high (1 mM) concentrations of added deoxynucleosides lend support to the notion of compartmentalized dNTP pools for repair and replication.     

Evidence for new cellular proteins which may negatively control DNA replication or cell growth in rat 3T3 fibroblasts

When separated and proliferating rat 3T3 cells are treated with butyrate (6 mM), DNA synthesis stops within 24 h, while RNA and protein synthesis proceed unaffected. This gradually converts normal cells into giant ones in the presence of butyrate (volume up to 30-fold greater). The giant cells stop growing when cell to cell contact is established. By studying the rate of synthesis of 300 cell proteins, we have identified two proteins (39 kDa, PI = 6.2, and 60 kDa, pI = 5.6) whose synthesis rises at least 10-fold when DNA replication and mitosis are prevented following intercellular contact or butyrate treatment, and another (64 kDa, pI = 5.6) whose synthesis rises at least 10-fold when cell growth stops by contact, both in the presence of butyrate and in the absence of butyrate (untreated confluent cells). The synthesis of some cellular oncogenes increases when the cell transits from G0 to S phase; the two proteins of 39 and 60 kDa described here are regulated in the opposite direction, their synthesis is enhanced when the cell leaves the proliferation cycle to enter G0.     

The effect of oestradiol on the DNA synthesis in neonatal mouse uterus and cervix

Summary (³H)-Thymidine autoradiography was used to study the DNA synthesis in the stroma and epithelium in the uterus proper and the uterine cervix of neonatal mice treated with oestradiol. It was found that in the epithelium of the uterus proper the DNA synthesis is stimulated between 6 and 12 h after injection of oestradiol and decreases again at 18 h. In the stroma of the uterus proper the DNA synthesis is increased 18 h after oestradiol injection.In the epithelium and stroma of the uterine cervix the DNA synthesis decreases from 5 h and is strongly depressed 18 h after oestradiol treatment.This work has been supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society (Landsforeningen mot Kreft).     

Ultraviolet irradiation inhibits encapsidation of simian virus 40 chromatin.

During normal maturation and majority of pulse-labeled simian virus 40 DNA progresses from chromatin to previrions and virions within 5 h. UV light inhibits this progression. In heavily irradiated cultures (108 J m-2) most of the simian virus 40 DNA synthesized immediately before irradiation remains as chromatin for at least 5 h. This inhibition of maturation seems to be a result of the inhibition of protein synthesis. The data suggest that the pool of proteins required for maturation is sufficient to convert one-third of the simian virus 40 DNA molecules labeled in a 10-min pulse (at 33 h postinfection) from chromatin to previrions and virions and is exhausted within 1 h.     

Origin activation requires both replicative and accessory helicases during T4 infection

The bacteriophage T4 has served as an in vitro model for the study of DNA replication for several decades, yet less is known about this process during infection. Recent work has shown that viral DNA synthesis is initiated from at least five origins of replication distributed across the 172 kb chromosome, but continued synthesis is dependent on recombination. Two proteins are predicted to facilitate loading of the hexameric 41 helicase at the origins, the Dda accessory helicase and the 59 loading protein. Using a real time, genome-wide assay to monitor replication during infections, it is shown here that dda mutant viruses no longer preferentially initiate synthesis near the origins, implying that the Dda accessory helicase has a fundamental role in origin selection and activation. In contrast, at least two origins function efficiently without the 59 loading protein, indicating that other factors load the 41 helicase at these loci. Hence, normal T4 replication includes two mechanistically distinct classes of origins, one requiring the 59 helicase loader, and a second that does not. Since both mechanisms require an additional factor, repEB, for sustained activation, normal T4 origin function appears to include at least three common elements, origin selection and initial activation, replisome loading, and persistence.     

Use of Fluorouracil-Uracil Combinations to Study Growth Accompanied by Insufficient Deoxyribonucleic Acid Synthesis in Bacillus cereus

5-Fluorouracil (FU) at a concentration of 16 muM almost totally inhibited deoxyribonucleic acid (DNA) synthesis and cell division by Bacillus cereus, whereas growth continued at an exponential rate (25% of control for at least 3 h). In cultures simultaneously given 160 muM uracil (U) along with the FU, DNA synthesis still stopped, but cell division continued for one generation at the control rate and at a much slower rate beyond that; in the meantime, cell mass continued to increase at an essentially normal rate. The cells in cultures treated with FU or FU plus U were elongated and contained about half of the control content of DNA, with one nuclear area per cell instead of two. Loss of cloning ability, unlike mass increase, was always correlated with the continuing inhibition of DNA synthesis, in either FU- or U plus FU-treated cultures.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.     

Stepwise effects of cytokinin activity and DNA synthesis upon mitotic cycle events in partially synchronized tobacco cells

Cytokinin addition to tobacco cell suspensions induced synchronous cell division after an 18 h lag period. Although continuous presence of the cytokinin in the culture medium during this lag period was essential to division, cytokinin was not required during mitosis itself. For each cell generation, cytokinin-dependent events are thus completed before mitosis occurs.Two experiments suggested that these cytokinin-dependent events are independent of DNA synthesis:

1. (i) With or without cytokinin, DNA synthesis proceeded normally in the presence of auxin, for at least the time required for one cell generation in complete medium.

2. (ii) In the presence of cytokinin, when DNA synthesis in the lag period was inhibited by FUdR, one normal cell division occurred when cytokinin was withdrawn and DNA synthesis restored by thymidine addition.

In cytokinin-starved cells, metaphase was greatly prolonged although prophase was unaffected.