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PubMed2000

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development.

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.

Autoradiography↗
PubMed1978

Recently replicated simian virus 40 DNA is a preferential template for transcription and replication.

The template activities for the processes of replication and transcription were compared for recently replicated ("new") and uniformly labeled ("old") simian virus 40 (SV40) DNA in infected monkey cells (line TC7). New SV40 DNA (pulse-labeled for 1 h) served as a template for a second round of replication with a relatively high probability (8% of the DNA replicated per h) for a period of 5 h, after which time its template activity rapidly decreased by severalfold. Old SV40 DNA (labeled for 24 h) functioned as a template for replication with a constant probability (1.8% of the DNA replicated per h) for at least 12 h. The proportion of RNA polymerase with nonreplicated and with recently replicated (bromodeoxyuridine-substituted) viral DNA was determined by an assay that used the Triton-soluble SV40 transcription complex. The proportion of RNA polymerase associated with nonreplicated SV40 DNA decreased very slowly (to 50% in 6 h), strongly suggesting that replicating viral genomes are not required as templates for the initiation of late transcription. This hypothesis was supported by the finding that the RNA synthesized in vitro was associated with covalently closed circular SV40 DNA. Furthermore, after 9 h in bromodeoxyuridine, the recently replicated viral DNA had nearly three times more RNA polymerase per unit of DNA than did the nonreplicated DNA. We thus conclude that recently replicated SV40 DNA is utilized preferentially as a template for transcription and for replication.

Cell Line↗
PubMed1989

Inhibition of c-myc gene expression in murine lymphoblastoma cells by geldanamycin and herbimycin, antibiotics of benzoquinoid ansamycin group.

We have shown that geldanamycin (GDM), an antibiotic of benzoquinoid ansamycin group, inhibits DNA replication in cultured mouse lymphoblastoma L5178Y cells. Here we report that GDM selectively inhibited the expression of c-myc gene, proto-oncogene, along with suppression of DNA replication in L5178Y cells, which are consistent with our previous results that c-myc protein promotes cellular DNA replication. The significantly enhanced inhibition by GDM of DNA replication was observed, when the antibiotic was introduced at G1 stage prior to S phase of cell cycle. The results are in favor of the prospects that GDM inhibits DNA replication mainly at time of initiation, and that c-myc protein is essential for the initiation of cellular DNA replication. Furthermore, when c-myc expression was inhibited by GDM, the expression of p53 gene, the product of which may be another DNA replication protein, was stimulated in the tumor cells. Thus, GDM should be useful to investigate the molecular mechanism of DNA replication promoted by c-myc protein and also to distinguish the function of c-myc protein from that of p53 protein in DNA replication.

Animals↗
PubMed1986

Sequential protooncogene expression during rat liver regeneration.

When growth is stimulated in the normally quiescent adult rat liver by partial hepatectomy, steady state levels of messenger RNAs (mRNAs) for c-fos, c-myc, and p53 increase sequentially during the prereplicative phase which precedes DNA synthesis. Levels of c-fos mRNA are elevated at least 4-fold within 15 min after partial hepatectomy and decrease rapidly by 2 h; c-myc mRNA reaches maximal levels (5-fold over normal) between 30 min and 2 h after the operation. A second, transient phase of expression for both c-fos and c-myc occurs around 8 h after partial hepatectomy. p53 mRNA levels increase between 8 and 12 h after the operation (5-fold over normal) and are reflected in an elevation of steady state levels of p53 protein between 12 and 15 h after partial hepatectomy. The levels of ras p21 protein increase much later at a time of active DNA replication and cell division. Actinomycin D injected at the time of partial hepatectomy blocks the increase in c-myc at 2 h but has no effect on c-fos mRNA levels. Actinomycin D injected at 6 h only partially blocks the increase in c-myc and p53 mRNA at 8 h but does not affect c-fos mRNA. Our results suggest that the transient and sequential expression of protooncogenes during the prereplicative stage of liver regeneration is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle and can serve as markers for identifying specific humoral factors involved in liver regeneration.

Albumins↗
PubMed2002

Control of replication timing by a transcriptional silencer.

BACKGROUND: Eukaryotic DNA replication starts at many origins. Some origins are used early in S phase, while others are programmed to fire later. In general, late replication is correlated with transcriptional inactivity and with location near the nuclear periphery. However, the mechanisms that determine replication timing are unclear, and the cause-and-effect relationship between late replication, transcriptional inactivity, and location at the nuclear periphery is unknown. RESULTS: Using budding yeast, we show that a transcriptional silencer, HMR-E, can reset the time of initiation of ARS305 from early to late. This resetting requires Sir proteins, which are silencers of transcription. Resetting can also be achieved by targeting Sir4 to ARS305. HMR-E sequences and targeted Sir4, both of which cause late replication of ARS305, also cause transcriptional silencing of the nearby APA1 gene. CONCLUSIONS: Sir proteins are sufficient to reprogram an origin from early to late; that is, Sir proteins are a cause of late replication. Presumably, the tight chromatin structure promoted by Sir proteins favors both transcriptional inactivity and late replication.

Base Sequence↗
PubMed1982

Late events in T4 bacteriophage DNA replication. III. Specificity of DNA reinitiation as revealed by hybridization to cloned genetic fragments.

Through the use of the technique of hybridization to cloned genes, the site specificity of the reinitiation of T4 DNA replication was examined at late times after infection, when a large amount of DNA had accumulated in the infected cell. Replication was examined under two conditions; (i) when there was recombination but the repair of the recombinants was inhibited, and (ii) when recombination was followed by covalent joining. When no covalent repair of recombinant was allowed, reinitiation occurred in the areas known to be also involved in the initiation of replication of the parental molecule: thus late reinitiation, if covalent joining is prevented, is site specific. When there was covalent joining, reinitiation displayed no apparent site specificity. The results are discussed in light of the possibility that at late times after infection recombinant intersections act as primers. The similarity of the model proposed to the "break-and-copy" model for lambda phage and the fitness of the proposed model to the genetic phenomena described by others are emphasized.

Base Sequence↗
PubMed1989

The beta subunit modulates bypass and termination at UV lesions during in vitro replication with DNA polymerase III holoenzyme of Escherichia coli.

The cycling time of DNA polymerase III holoenzyme during replication of UV-irradiated single-stranded (ss) DNA was longer than with unirradiated DNA (8 versus 3 min, respectively), most likely due to slow dissociation from lesion-terminated nascent DNA strands. Initiation of elongation on primed ssDNA was not significantly inhibited by the presence of UV lesions as indicated by the identical distribution of replication products synthesized at early and late reaction times and by the identical duration of the initial synthesis bursts on both unirradiated and UV-irradiated DNA templates. When replication was performed with DNA polymerase III* supplemented with increasing quantities of purified beta 2 subunit, the cycling time on UV-irradiated DNA decreased from 14.8 min at 1.7 nM beta 2 down to 6 min at 170 nM beta 2, a concentration in which beta 2 was in large excess over the polymerase. In parallel to the reduction in cycling time, also the bypass frequency of cyclobutane-photodimers decreased with increasing beta 2 concentration, and at 170 nM beta 2, bypass of photodimers was essentially eliminated. It has been shown that polymerase complexes with more than one beta 2 per polymerase molecule were formed at high beta 2 concentrations (Lasken, R. S., and Kornberg, A. (1987) J. Biol. Chem. 262, 1720-1724). It is plausible that polymerase complexes obtained under high beta 2 concentration dissociate from lesion-terminated primers faster than polymerase complexes formed at a low beta 2 concentration. This is expected to favor termination over bypass at pyrimidine photodimers and thus decrease their bypass frequency. These results suggest that the beta 2 subunit might act as a sensor for obstacles to replication caused by DNA damage, and that it terminates elongation at these sites by promoting dissociation. The intracellular concentration of beta 2 was estimated to be 250 nM (Kwon-Shin, O., Bodner, J. B., McHenry, C. S., and Bambara, R. A. (1987) J. Biol. Chem. 262, 2121-2130) and is 15-fold higher than the estimated intracellular concentration of DNA polymerase III holoenzyme (15 nM). This high concentration of beta 2 may be responsible for the observation that very little (if any) bypass of pyrimidine photodimers occurred in vivo when the SOS system was not induced. Moreover, it predicts that bypass synthesis under SOS conditions might be associated with an altered form of the beta subunit.

Bacteriophages↗
PubMed1984

[DNA repair in Drosophila oogenesis].

In experiments with females of lines with an impaired DNA repair systems mei-9 (impaired excision repair) and mei-41 (impaired postreplicative repair), a method of successive irradiation by X-rays (1000 R) and hyperthermia (+37 degrees C) action was used for the purpose of defining a moment when DNA repair takes place in oogenesis. Repair in mature mei-41 oocytes judged of by synergism effect of the both factors acting was ascertained to take place right after X-raying (prior to DNA replication) and being absent at the fertilization period (at the time of or after DNA replication). DNA repair in mei-9 females was not registered in both cases. On the basis of these facts, it is suggested that coordination of various DNA repair systems is necessary for damaged chromosomes to be repaired. It is also concluded that the method used can be regarded as an effective technique in the study of mutation process.

Animals↗
PubMed1999

Telomeric chromatin modulates replication timing near chromosome ends.

Saccharomyces cerevisiae telomeric DNA replicates late in S phase, and telomeric genes are transcriptionally silent. Transcriptional repression of telomere-proximal genes results from silent chromatin initiating at the chromosome end, but the relationship between telomeric chromatin and DNA replication is unknown. Mutations in SIR3, a silent chromatin component, cause telomeric DNA on chromosome V to replicate much earlier because of earlier initiation of a nearby replication origin, the Y' ARS. A second telomere-proximal ARS, from an X element, does not act as an origin in a wild-type strain, whereas in a sir3 cell it does. We conclude that telomeric chromatin has a Sir3-dependent inhibitory effect on DNA replication.

Chromatin↗
PubMed1984

Replication timing of genes and middle repetitive sequences.

DNA replication in mammals is temporally bimodal. "Housekeeping" genes, which are active in all cells, replicate during the first half of the S phase of cell growth. Tissue-specific genes replicate early in those cells in which they are potentially expressed, and they usually replicate late in tissues in which they are not expressed. Replication during the first half of the S phase is, therefore, a necessary but not sufficient condition for gene transcription. A change in the replication timing of a tissue-specific gene appears to reflect the commitment of that gene to transcriptional competence or to quiescence during ontogeny. Most families of middle repetitive sequences replicate either early or late. These data are consistent with a model in which two functionally distinct genomes coexist in the nucleus.

Animals↗
PubMed1977

Chromatin replication in vitro. Properties of a HeLa nuclear system.

An in vitro HeLa chromatin replication system was developed and characterized. Purified nuclei synthesized DNA from replication forks which were undergoing chain elongation at the time of cell fractionation. DNA replication in isolated nuclei was deficient in DNA maturation functions; the Okazaki fragments synthesized in vitro accumulated and were ligated to high-molecular-weight DNA with low efficiency. The chromatin protein components of nuclei incubated in the DNA replication assay conditions were investigated for displacement, degradation, and exchange. Displacement of total nuclear protein during in vitro incubation occurred to the extent of 3%, and involved only the nonhistone nuclear proteins. Degradation was not detectable, assayed both by loss of acid-soluble radioactivity and by protein electrophoretic patterns in polyacrylamide gels. No detectable protein was exchanged from chromatin to exogenous DNA.

Cell Nucleus↗
PubMed1982

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation.

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G0 state. Autoradiography studies of cells released from G0 after 72 h and replated at lower densities (3-9 x 10(3) cells/cm2) in fresh medium containing 15% fetal bovine serum showed that semi-conservative DNA synthesis (S phage) began approximately 24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G0, allowed them to reattach at lower densities, irradiated them in early G1 (approximately 18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G0, plated at cloning densities and irradiated as soon as they had attached and flattened out. (approximately 20 h prior to S) or 4, 8, 12, 16, 20, or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.

Cell Line↗
PubMed1995

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis.

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.

Bacillus subtilis↗
PubMed1979

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome.

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.

Animals↗
PubMed1993

Sequence of centromere separation. Minor satellite DNA does not influence separation of inactive centromeres in transformed cells of mouse.

Neoplastic cells may carry inactive centromeres on some multicentric, yet stable, chromosomes. We report that some inactive centromeres in L929 mouse cells do not contain minor satellite DNA, the DNA fraction which has been suggested to constitute the centromere. We compared the sequence of separation of inactive centromeres carrying the minor satellite with those lacking this fraction. The sequence of separation appears to be independent of whether or not the inactive centromeres carry the minor satellite DNA. The timing of replication of the inactive centromeres is also independent of this DNA. Hence, minor satellite of mouse is not a factor in holding together the subunits of inactive centromeres. Extension of these results to active centromeres might suggest that the minor satellite DNA is not a factor responsible for adhesion of the two centromere sub-units up until late meta-anaphase.

Animals↗
PubMed1970

The dampening of DNA replication cycles after x-irradiation or ultraviolet irradiation.

Escherichia coli strains 15T(-) (555-7) and B/r were grown in the presence of thymine-(14)C to label all DNA. The ability of these parental DNA's to undergo cycles of replication subsequent to cellular irradiation with either X-ray or ultraviolet light (UV) was followed with density labels. Exposed cells were shifted into the density medium at times which were approximately multiples of normal rounds of DNA replication. A portion of the parental DNA, replicated semiconservatively once during an initial cycle following UV or X-irradiation in E. coli, failed to replicate again within the time studied. The time course of semiconservative parental DNA replication is altered.

Carbon Isotopes↗