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Results for “DNA Replication Timing”

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PubMed1981

Evidence of diurnal variation of human epidermal cell proliferation. II. Duration of epidermal DNA synthesis.

In the epidermis of six healthy male volunteers the DNA synthesis time was measured by in vitro double labeling technique in a longitudinal study (48 h) with taking skin punch biopsies every 6 h. The external conditions of the experiments and one group of volunteers were identical to those of a previous study on LI of the epidermal keratinocytes. 14C-thymidine and 3H-thymidine were used for double labeling the DNA-synthesizing nuclei of keratinocytes. The duration of DNA replication time proved to be nearly constant, with minor fluctuation, during the entire time span of experiments (mean 6.19 +/- 0.3 h). Thus, the present results are in accordance with the conception that a periodically changing entrance rate of G1-cells into the S-phase of the generation cycle of keratinocytes is predominantly responsible for circadian variations in the number of DNA-synthesizing epidermal keratinocytes.

Adult↗
PubMedDate not supplied

C-myc transcript is induced in rat liver at a very early stage of regeneration or by cycloheximide treatment.

In rats, partial hepatectomy induces reasonably synchronized DNA replication in the remaining liver after approximately 20 h. Events occurring during the earlier stages of liver regeneration are of interest because they may tell us how cells in vivo respond when they move from a differentiated resting state (G0 phase) to a proliferative state. We report here that the expression of the c-myc oncogene is increased up to 10-15-fold of the normal level within 1-3 h after partial hepatectomy. This expression begins to decrease rapidly after 4 h and has returned to less than double the normal level after 8 h, at which time replicative DNA synthesis has still not begun. A still larger increase in c-myc transcription (approximately 600-fold) is observed in the liver when protein synthesis is inhibited by an injection of cycloheximide. These findings suggest the existence of a short-lived protein that is synthesized soon after partial hepatectomy, and which suppresses the expression of c-myc.

Animals↗
PubMed1987

Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells.

We have critically evaluated various modifications of the alkaline elution methodology that were required to adapt the method for measuring DNA damage in cells from animal tissues treated in vivo. These modifications involved the use of a fluorometric assay for the eluted DNA using the dye Hoechst 33258, which in turn required the use of a different combination of filter and lysis conditions than those used in conventional assays. This protocol was compared with the conventional protocols by examining the DNA damage produced in cultured Chinese hamster ovary cells after treatment with three agents (gamma-rays, cis-dichlorodiammineplatinum (DDP) and trans-DDP) that differ widely in the type and repairability of the DNA lesions that they induce. For both gamma-rays and trans-DDP, the results obtained by the various protocols were equivalent with respect to the amount, type, and rate of repair of the DNA damage produced. On the other hand, for cis-DDP, where the repair time for DNA crosslinks was significantly long relative to the cell-cycle time, DNA replication appeared to be a potentially complicating factor in the measurement of crosslink repair. However, even after treatment of rapidly dividing cultured cells, where any discrepancy between the radioactivity and Hoechst assays due to DNA replication should be maximal, the resulting difference in the amount of repair measured using the two assays was relatively small. Finally, in experiments using cis-DDP and trans-DDP, the data suggested that when polycarbonate and polyvinyl chloride filters were compared using the same cell lysis conditions, their relative sensitivity to detect DNA-protein versus DNA-interstrand crosslinking were comparable. The modified alkaline elution protocol for the measurement of DNA damage in vivo therefore appears, in most cases, to produce results comparable with those obtained by the conventional protocols.

Animals↗
PubMed2026

Genetic control of local mutation rates.

Mutations are the source of evolutionary novelty but also the cause of genetic diseases and cancer. Mutation rates are known to be heterogeneous along the genome, however the extent to which local mutation rates vary among individuals in a population and are genetically determined is unknown. To test this, we analyzed the chromosomal distribution of somatic mutations in cell lines from 1,662 individuals, controlling for the confounding effects of DNA replication timing on local mutation rates and of trans-acting modulators on global mutation rates. We describe substantial interindividual variation in mutation rates across the human genome. By comparing mutation-rate variation to individuals' genotypes, we identified 35 instances in which polymorphic alleles in the population associate with somatic mutation rates in their vicinity. We call these mutation quantitative trait loci (mutQTLs). mutQTLs associated with somatic mutations in lymphoblastoid cell lines and in chronic lymphocytic leukemia, and with germline genetic variants. Two of the four mutQTLs inferred to be associated with germline mutation-rate variation were located within large clusters of zinc-finger genes and transposable elements, where they functioned as cis-mutators conferring an increased rate of mutation in their vicinity. mutQTLs provide a portal into the evolution of mutation rate heterogeneity across the genome and across individuals.

Humans↗
PubMed1992

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

Bacterial Proteins↗
PubMed1989

Methylation of GATC sites is required for precise timing between rounds of DNA replication in Escherichia coli.

We have used the Koppes and Nordstrøm (Cell 44:117-124, 1986) CsCl density transfer approach for analysis of DNA from exponentially growing, isogenic Escherichia coli dam+ and dam mutant cells to show that timing between DNA replication initiation events is precise in the dam+ cells but is essentially random in the dam cells. Thus, methylation of one or more GATC sites, such as those found in unusual abundance within the origin, oriC, is required for precise timing between rounds of DNA replication, and precise timing between initiation events is not required for cell viability. Both the dam-3 point mutant and the delta(dam)100 complete deletion mutant were examined. The results were independent of the mismatch repair system; E. coli mutH cells showed precise timing, whereas timing in the isogenic E. coli mutH delta(dam)100 double mutant was random. The mechanism is thus different from the role of Dam methylation in mismatch repair and probably involves conversion of hemimethylated GATC sites present in daughter origins just after initiation to a fully methylated state.

DNA Replication↗
PubMed1983

High resolution analysis of the timing of replication of specific DNA sequences during S phase of mammalian cells.

A new method, utilizing selective photodegradation of 5-bromo-deoxyuridine (BUdR)-substituted DNA and flow cytometry, has been developed for analyzing the timing of replication of specific DNA sequences. Chemically synchronized Chinese hamster ovary cells were given a pulse of the deoxythymidine analogue, BUdR, at different times during S phase, and flow sorted according to DNA content, before DNA isolation. Newly-replicated, unifilarly BUdR-substituted DNA was selectively degraded by treatment with 33258 Hoechst plus near UV light followed by S1 nuclease digestion; the resistant DNA was analyzed for its content of 18s and 28s rDNA or dihydrofolate reductase (DHFR) sequences via Southern blot analysis. Both the rDNA and DHFR sequences were found to replicate almost entirely during the first quarter of S phase. The approach described should have general utility for analyzing replication kinetics of specific DNA sequences in mammalian cells.

Animals↗
PubMed1987

Periodic synthesis of phospholipids during the Caulobacter crescentus cell cycle.

Net phospholipid synthesis is discontinuous during the Caulobacter crescentus cell cycle with synthesis restricted to two discrete periods. The first period of net phospholipid synthesis begins in the swarmer cell shortly after cell division and ends at about the time when DNA replication initiates. The second period of phospholipid synthesis begins at a time when DNA replication is about two-thirds complete and ends at about the same time that DNA replication terminates. Thus, considerable DNA replication, growth, and differentiation (stalk growth) occur in the absence of net phospholipid synthesis. In fact, when net phospholipid synthesis was inhibited by the antibiotic cerulenin through the entire cell cycle, both the initiation and the elongation phases of DNA synthesis occurred normally. An analysis of the kinetics of incorporation of radioactive phosphate into macromolecules showed that the periodicity of phospholipid synthesis could not have been detected by pulse-labeling techniques, and only an analysis of cells prelabeled to equilibrium allowed detection of the periodicity. Equilibrium-labeled cells also allowed determination of the absolute amount of phosphorus-containing macromolecules in newborn swarmer cells. These cells contain about as much DNA as one Escherichia coli chromosome and about four times as much RNA as DNA. The amount of phosphorus in phospholipids is about one-seventh of that in DNA, or about 3% of the total macromolecular phosphorus.

Cell Cycle↗
PubMed1976

Prolongation of replication time after doublings of the DNA content of polytene chromosome bands of Chironomus.

Using 3H-thymidine autoradiography, labeling frequency of homologous asynapsed chromosome bands of the hybrid of Chironomus th. thummi and Chironomus th. piger has been studied. In a number of these bands the DNA content of the thummi bands if 2, 4, 8 or 16 times as large as that of the homologous piger bands (Keyl, 1965). Those bands of CH. TH. thummi which show one doubling of their DNA content in comparison with the homologous piger bands are also labeled two times more frequently than piger. In contrast to this such a correlation between increase of labeling frequency (i.e. prolongation of replication time) and doubling of the DNA content is not observed, when thummi bands have 4, 8 or 16 times more DNA than their homologues in piger. In these cases replication time is also prolonged after each doubling. Duration of DNA synthesis increases linearly but always by a smaller factor as the corresponding DNA content is increased.

Animals↗
PubMed2025

Regulation of replication timing in Saccharomyces cerevisiae.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Saccharomyces cerevisiae↗
PubMed1983

Cell cycle time and possible early DNA replication in C-band regions in the domestic pig (Sus scrofa) lymphocytes.

Dynamic cell replication studies on peripheric blood lymphocytes cultured from the domestic pig (Sus scrofa) were performed. Replication started after 18-24 hr in culture and the cell cycle time was about 8-10 hr. Chromosomes from cells at first division showed C-banded-like regions. This finding suggests the occurrence of early DNA replication zones within the constitutive heterochromatin of the pig chromosomes.

Animals↗
PubMed1982

DNA replication in short-time organ cultures of human epidermis. Inhibition by aphidicolin, and detection of DNA polymerases alpha, beta, and gamma.

Human epidermis uncontaminated by fibroblasts was isolated by a suction blister method. DNA synthesis in short-time organ cultures of isolated epidermis was strongly inhibited by aphidicolin, suggesting that DNA polymerase alpha is involved in DNA replication in human epidermis. On the basis of their responses to inhibitors, primer-template requirements, and chromatographic properties, DNA polymerases alpha, beta, and gamma were all identified in epidermal extracts.

Aphidicolin↗
PubMed1987

Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication.

Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.

Animals↗
PubMed1976

Timing of nucleolar DNA replication in Amoeba proteus.

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.

Amoeba↗
PubMed1990

DNA contents of replication without DNA density labeling.

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.

Cell Cycle↗
PubMed1988

Asynchronous DNA replication within the human beta-globin gene locus.

The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.

Cell Cycle↗