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PubMed1982

Sodium butyrate stimulates DNA repair in UV-irradiated normal and xeroderma pigmentosum human fibroblasts.

Histone acetylation, DNA replicative synthesis, UV-induced DNA repair synthesis, and UV-induced endonuclease-sensitive sites were measured in normal human fibroblasts and xeroderma pigmentosum fibroblasts (complementation groups A, C, and D) following exposure to sodium butyrate. In all four cell types, treatment with millimolar concentrations of sodium butyrate resulted in a hyperacetylation of the core histones. Furthermore, following an exposure of 20 mM sodium butyrate for 48 h, the extent of hyperacetylation was the same in each cell type. In agreement with previous reports, we observed a marked decrease in DNA replicative synthesis in each cell type following increasing times of exposure to sodium butyrate. On the other hand, we observed a marked increase in DNA repair synthesis occurring during early times after UV irradiation in normal cells and in two of the xeroderma pigmentosum cell strains (groups C and D). This increase appeared to correlate with the increase in the highest acetylated form of histone H4. Furthermore, the total number of endonuclease-sensitive sites (i.e. prior to the onset of repair) induced by UV radiation was the same in both butyrated-treated and untreated normal cells over the dose range of 0-20 J/m2. However, the initial rate of removal of these sites increased in butyrate-treated normal cells. These results indicate that sodium butyrate stimulates the initial rate of nucleotide excision repair in both normal and (partially) repair-deficient human cells at concentrations where the histones are maximally hyperacetylated.

Butyrates↗
PubMed1987

Interaction between the DNA polymerase and single-stranded DNA-binding protein (infected cell protein 8) of herpes simplex virus 1.

The herpes virus-encoded DNA replication protein, infected cell protein 8 (ICP8), binds specifically to single-stranded DNA with a stoichiometry of one ICP8 molecule/12 nucleotides. In the absence of single-stranded DNA, it assembles into long filamentous structures. Binding of ICP8 inhibits DNA synthesis by the herpes-induced DNA polymerase on singly primed single-stranded DNA circles. In contrast, ICP8 greatly stimulates replication of circular duplex DNA by the polymerase. Stimulation occurs only in the presence of a nuclear extract from herpes-infected cells. Appearance of the stimulatory activity in nuclear extracts coincides closely with the time of appearance of herpes-induced DNA replication proteins including ICP8 and DNA polymerase. A viral factor(s) may therefore be required to mediate ICP8 function in DNA replication.

Bacteriophages↗
PubMed1994

Peripheral benzodiazepine stimulates secretion of growth hormone and mitochondrial proliferation in pituitary tumour GH3 cells.

High affinity receptors for peripheral benzodiazepines (PBD) are present within the pituitary gland. Whether these receptors influence cell growth, mitochondrial morphology and secretion of growth hormone was examined in pituitary tumour GH3 cells. Cells were incubated in medium with either 10% calf serum, or serum free medium, or serum free medium plus 10 nM of selective PBD ligands (PK11195 or Ro5-4864) or a central benzodiazepine ligand (clonazepam). Peripheral or central ligands had no effect on cell growth. Quantitative electron micrography, however, revealed that cells treated with PBDs had a 2.5-fold increase in the total mitochondrial area within cells, a 1.6-fold increase in the number of mitochondria, and a 2.0 to 2.7-fold increase on the number of dividing mitochondria compared to serum free controls. The activity of DNA polymerase gamma, which replicates mitochondrial DNA, was increased approximately 3 times the initial value after 6 h exposure to PBDs. The number of nucleolar organizer regions (NORs), which relate to the ultimate synthesis of cellular protein, showed a 1.4-fold increase in PBDs treated cells. Secretion of growth hormone was stimulated (240% of serum free control) by exposure to PBDs for 12 h. The central benzodiazepine ligand, clonazepam, had little effect on mitochondrial morphology or secretion of growth hormone. These findings suggest that PBD receptors may be involved in mitochondrial proliferation and may affect the secretion of pituitary hormones.

Adenoma↗
PubMed1968

Regulation and timing of deoxyribonucleic acid synthesis in hyphae of Aspergillus nidulans.

Pulse labeling of deoxyribonucleic acid (DNA) and radioautography have been used to study the effect of growth rate on nuclear replication in Aspergillus nidulans. When conidia were germinated in media supporting a fast growth rate, the radioactive pulse labeled either all of the nuclei in a cell or none of them. At slower growth rates, hyphae contained both labeled and unlabeled nuclei. Altering the growth rate thus changed nuclear replication from simultaneous to sequential. The time taken to duplicate the DNA in a nucleus, estimated from the ratio of labeled to total nuclei, remained constant at the different doubling times. The distribution of label showed that nuclei in the same hypha spent unequal times in both the postmitotic gap (G1) and the premitotic gap (G2) periods when grown at slow rates. These unequal G1 and G2 periods are considered to cause asynchrony. Once DNA synthesis was out of phase through growth on a poor medium, transferring the hypha to a rich medium did not resynchronize the nuclei. To interpret the data, two initiator mechanisms, one starting DNA synthesis and the other mitosis, are postulated to control nuclear replication in A. nidulans.

Adenine↗
PubMed1974

The relative numbers of different genes in exponential microbial cultures.

It is shown that the results of the marker frequency analysis of Sueoka and Yoshikawa (1965) can be derived as very good approximations from a model where the rigid assumptions of their analysis are relaxed to take into account statistical variations in the timing of cell events. It is further shown that the expression for the amount of DNA per cell can be approximated by an elementary exponential function of the growth rate, and this result facilitates genetic mapping by DNA hybridization techniques. An analysis of recent data on gene frequencies in Escherichia coli corroborates a model of symmetric, bidirectional chromosome replication with a replication time of approximately thirty minutes.

Cell Division↗
PubMed1995

Transition in specification of embryonic metazoan DNA replication origins.

In early Xenopus embryos, in which ribosomal RNA genes (rDNA) are not transcribed, rDNA replication initiates and terminates at 9- to 12-kilobase pair intervals, with no detectable dependence on specific DNA sequences. Resumption of ribosomal RNA (rRNA) synthesis at late blastula and early gastrula is accompanied by a specific repression of replication initiation within transcription units; the frequency of initiation within intergenic spacers remains as high as in early blastula. These results demonstrate that for rRNA genes, circumscribed zones of replication initiation emerge in intergenic DNA during the time in metazoan development when the chromatin is remodeled to allow gene transcription.

Animals↗
PubMed1995

Factors affecting the timing and imprinting of replication on a mammalian chromosome.

Fluorescence in situ hybridisation has been used to follow replication of the short arm of human chromosome 11 using chromosome anomalies to distinguish the maternally-and paternally-derived homologues. The temporal difference in replication timing within and between chromosomes has been estimated by combining S phase detection with dual colour fluorescence in situ hybridisation. Proximal regions of 11p, including the WT1 gene, tend to replicate earlier on the maternally-derived chromosome than on the paternally-derived homologue. More distal parts of 11p (including the IGF2 gene) have the opposite imprint. The average difference in replication timing between homologous loci in the population of cells is small compared to the differences between loci along a single chromosome. The imprint is not strictly adhered to since many nuclei have hybridisation patterns opposite to the trend within the population. The nature of the imprinting signal has been investigated. Absolute replication time, but not the imprint, was affected by azacytidine, an inhibitor of DNA methylation. The replication imprint was modified by treatments that inhibit histone deacetylation. We suggest that replication imprinting reflects differences in chromatin structure between homologues.

Chromosome Aberrations↗
PubMed1977

Characterization of simian virus 40 tsA58 transcriptional intermediates at restrictive temperatures: relationship between DNA replication and transcription.

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.

Cell Line↗
PubMed1998

Ordered expression of ftsQA and ftsZ during the Caulobacter crescentus cell cycle.

The mechanisms by which bacterial cell division and DNA replication are co-ordinated are still unknown. We have used the easily synchronizable bacterium Caulobacter crescentus to determine when the cell division genes ftsQ and ftsA are transcribed during the DNA replication cycle and to compare their transcription with that of ftsZ. Unlike the situation in Escherichia coli, transcription of ftsQ and ftsA does not extend into ftsZ in Caulobacter. ftsQ and ftsA are co-transcribed by a strong promoter, P(QA), present within the end of the ddl gene upstream of ftsQ. Transcription of P(QA) is turned on at the end of the DNA replication period, coincident with the end of the ftsZ transcription period. ftsA is also transcribed by another promoter, P(A), present between ftsQ and ftsA. P(A) transcription is approximately 10 times weaker than P(QA) and occurs during the DNA replication period. Transcription of ftsA by P(A) is sufficient for cell viability, but is not sufficient for normal cell division. When the transcription of ftsA is increased constitutively, cell division is inhibited and stalks are synthesized at aberrant positions. Thus, transcription of ftsA and ftsZ mimics their order of action in Caulobacter and proper transcription of ftsA has to be maintained for normal cell division and differentiation.

Amino Acid Sequence↗
PubMed1975

Effects of caffeine on postreplication repair in xeroderma pigmentosum cells.

Compared to its effects on unirradiated cells, caffeine inhibited DNA chain elongation and joining in UV-irradiated xeroderma pigmentosum (XP) cells but not in irradiated normal human cells. The drug also inhibited the ability of XP cells to recover and make DNA of normal size at long times after irradiation.

Caffeine↗
PubMed1994

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.

ATP-Binding Cassette Transporters↗
PubMed2003

Life history and the male mutation bias.

If DNA replication is a major cause of mutation, then those life-history characters, which are expected to affect the number of male germline cell divisions, should also affect the male to female mutation bias (alpha(m)). We tested this hypothesis by comparing several clades of bird species, which show variation both in suitable life-history characters (generation time as measured by age at first breeding and sexual selection as measured by frequency of extrapair paternity) and in alpha(m), which was estimated by comparing Z-linked and W-linked substitution rates in gametologous introns. Alpha(m) differences between clades were found to positively covary with both generation time and sexual selection, as expected if DNA replication causes mutation. The effects of extrapair paternity frequency on alpha(m) suggests that increased levels of sexual selection cause higher mutation rates, which offers an interesting solution to the paradox of the loss of genetic variance associated with strong directional sexual selection. We also used relative rate tests to examine whether the observed differences in alpha(m) between clades were due to differences in W-linked or Z-linked substitution rates. In one case, a significant difference in alpha(m) between two clades was shown to be due to W-linked rates and not Z-linked rates, a result that suggests that mutation rates are not determined by replication alone.

Age Factors↗
PubMed1997

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition.

Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26-27, 11p13, 11p15.5 and 15q11.2-12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2'-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication.

Adult↗
PubMed1984

Induction of polyploid nuclei in the plasmodium of Physarum polycephalum by platinum antitumor compounds.

The intranuclear mitosis of the plasmodial nuclei of myxomycetes permits the observation of defects in chromosomal repartition which would probably be lethal in other eukaryotic cells with open mitosis. We found that antitumoral platinum-amine compounds perturbed late mitotic events and induced the formation of giant nuclei which were polyploid in plasmodia of Physarum polycephalum. Using 26 platinum-amine complexes, we have shown that all antitumoral compounds induced the formation of polyploid nuclei for drug concentrations at least three times lower than the amount necessary to block the overall plasmodial growth, whereas platinum compounds without antitumor activity did not behave this way. DNA replication appeared to be quantitatively normal during formation of giant nuclei by antitumoral compounds. These observations suggest that platinum-amine compounds exert their antitumor activity by interfering with mitosis rather than by a gross inhibition of DNA synthesis.

Cell Nucleolus↗
PubMed2005

Identification of chondrocyte proliferation following laser irradiation, thermal injury, and mechanical trauma.

BACKGROUND AND OBJECTIVE: Cartilage has a limited regenerative capacity, and there are a lack of reliable techniques and methods to stimulate growth of new tissue to treat degenerative diseases and trauma. This study focused on identifying chondrocyte cell proliferation in ex vivo cartilage tissue following heating Nd:YAG laser using whole-mount analysis and flow cytometry, and compared findings with results produced by contact, and water bath heating methods, mechanical injury, and the addition of transforming growth factor-beta (TGF-beta). STUDY DESIGN/MATERIALS AND METHODS: Ex vivo rabbit nasal septal cartilages were either irradiated with an Nd:YAG laser (lambda = 1.32 microm, 2-16 seconds, 6 W/cm(2)), heated by immersion in a warm saline bath, heated by direct contact with a metal rod, or mechanically damaged by scoring with a scalpel or crushing. After treatment, specimens were incubated for 7 or 14 days in growth media containing 10 microM bromodeoxyuridine (BrdU). Additional specimens were cultured with both BrdU and TGF-beta. Both whole-mount BrdU-double-antibody detection techniques and flow cytometry were used to determine the presence of DNA replication as a marker of proliferation. RESULT: An annular region of regenerating chondrocytes was identified surrounding the laser irradiation zone in whole-mount tissue specimens, and the diameter of this region increased with irradiation time. Using whole-mount analysis, no evidence of chondrocyte DNA replication was observed in tissues heated using non-laser methods, grown in TGF-beta, or mechanically traumatized. In contrast, flow cytometry identified the presence of BrdU-positive cells in the S-phase of the cell cycle (synthesis of DNA) for all protocols, indicating chondrocyte proliferation. The percentage of cells that are in S-phase increased with irradiation time. CONCLUSION: These data provide evidence that laser irradiation, along with other thermal and mechanical treatments, causes a proliferative response in chondrocytes, and this is observed ex vivo in the absence of cellular and humoral repair mechanisms. The advantage of using optical methods to generate heat in cartilage is that microspot injuries could be created in tissue and scanned across surfaces in clinical applications.

Animals↗
PubMed2001

ATP utilization by yeast replication factor C. IV. RFC ATP-binding mutants show defects in DNA replication, DNA repair, and checkpoint regulation.

Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFC genes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1, RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in all RFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 and RFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the rfc1-K359R and rfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function of RFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation.

Adenosine Triphosphate↗
PubMed1999

A phenotype for enigmatic DNA polymerase II: a pivotal role for pol II in replication restart in UV-irradiated Escherichia coli.

DNA synthesis in Escherichia coli is inhibited transiently after UV irradiation. Induced replisome reactivation or "replication restart" occurs shortly thereafter, allowing cells to complete replication of damaged genomes. At the present time, the molecular mechanism underlying replication restart is not understood. DNA polymerase II (pol II), encoded by the dinA (polB) gene, is induced as part of the global SOS response to DNA damage. Here we show that pol II plays a pivotal role in resuming DNA replication in cells exposed to UV irradiation. There is a 50-min delay in replication restart in mutant cells lacking pol II. Although replication restart appears normal in DeltaumuDC strains containing pol II, the restart process is delayed for >90 min in cells lacking both pol II and UmuD'(2)C. Because of the presence of pol II, a transient replication-restart burst is observed in a "quick-stop" temperature-sensitive pol III mutant (dnaE486) at nonpermissive temperature. However, complete recovery of DNA synthesis requires the concerted action of both pol II and pol III. Our data demonstrate that pol II and UmuD'(2)C act in independent pathways of replication restart, thereby providing a phenotype for pol II in the repair of UV-damaged DNA.

Bacterial Proteins↗
PubMed1968

The centriole cycle in synchronized HeLa cells.

Progression of the HeLa cell through its life cycle is accompanied by centriolar replication and pericentriolar changes that are in synchrony with DNA synthesis and mitosis. The first signs of preparation for replication occur during G(1) at which time the two orthogonal centrioles separate. Replication by budding begins at/or near the initiation of DNA synthesis and is completed by G(2). Pericentriolar changes which probably are causally related to spindle tubule formation occur at this time and include the appearance of vesicles, electron-opaque bodies, and an amorphous pericentriolar halo. These phenomena begin to disappear by late prophase, and the remainder of mitosis manifests decreasing centriolar and pericentriolar activity.

Carbon Isotopes↗