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PubMed1990

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

Chromatin↗
PubMed1975

Replication of human chromosomes in human-mouse hybrids: evidence that the timing of DNA synthesis is determined independently in each human chromosome.

The terminal phase of DNA replication was studied by autoradiography in hybrids between human lymphocytes and mouse fibroblasts. The hybrids contained on the average only 11 human chromosomes. It was found that the sequence of terminal DNA replication for the human chromosomes in the hybrids was the same as the sequence of terminal replication for the corresponding chromosomes in the human lymphocytes. Furthermore, it was shown that the maintenance of the normal terminal replication sequence of the human chromosomes in the hybrids was not dependent on the presence of any specific human chromosome. The results suggest that the timing of terminal DNA replication is determined independently in each human chromosome.

Animals↗
PubMed1995

Replication through the terminus region of the Bacillus subtilis chromosome is not essential for the formation of a division septum that partitions the DNA.

Germinated and outgrowing spores of a temperature-sensitive DNA initiation mutant of Bacillus subtilis were allowed to initiate a single round of replication by being shifted from 34 to 47 degrees C at the appropriate time. The DNA replication inhibitor 6-(parahydroxyphenylazo)-uracil was added to separate portions of the culture at various times during the round. Samples were collected from each around the time of the first division septation for measurements of the extent of the round completed, the level of division septation, the position of the septum within the outgrown cell, and the distribution of DNA (nucleoid) in relation to the septum. The extent of replication was measured directly through a hybridization approach. The results show clearly that a central division septum can close down onto a chromosome that is only partially replicated (to a minimum extent of about 60% of the round) such that DNA appears on both sides of the septum and frequently very close to it. It is concluded, as claimed previously on the basis of a less direct approach (T. McGinness and R.G. Wake, J. Mol. Biol. 134:251-264, 1979), that replication through the terminus region of the chromosome is not essential for the formation of a division septum that partitions the DNA.

Bacillus subtilis↗
PubMed1980

Analysis of a cell cycle model for Escherichia coli.

Ribosome and protein synthesis, DNA replication and cell division in Escherichia coli cells are described by a mathematical model that integrates previous descriptions in quantitative terms and proposes a new formalization to relate ribosome net synthesis to cell growth. The model assumes a cell size control of DNA replication and therefore is structurally divided into two subsystems: the first, whose state variables are ribosomes and protein, and the second, which is activated when the protein level reaches a threshold and which is comprised of DNA replication and cell division. The dynamics of the entire system is set only by the first subsystem: the values of its parameters determine whether the cells will be in a resting condition or will grow exponentially and in the latter case the resulting duplication time, while the structure and the parameter values of the second subsystem determine the size and the composition of the cell and the timing of DNA replication during the cycle. Relationships are derived that allow a simple determination of the time of initiation and of termination of DNA replication and the number of chromosome origins involved in any possible cell cycle as well as the macromolecular levels at the beginning of a cycle and on the average in a population of cells in balanced exponential growth.

Animals↗
PubMed1998

Parental alleles of an imprinted mouse transgene replicate synchronously.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

Alleles↗
PubMed1992

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.

Animals↗
PubMed1998

A novel drug screening assay for papillomavirus specific antiviral activity.

Discovery and development of human papillomavirus (HPV) specific antiviral agents have been hampered by the lack of an in vitro assay permissive to HPV replication. An experimental assay system for monitoring HPV-11 DNA replication has been optimized for use as a papillomavirus antiviral drug screening tool. Cloned HPV DNA was introduced into SCC-4 cells by electroporation and viral DNA replication monitored by Southern blot. Kinetic studies demonstrated an increased HPV genome copy number with time. Viral DNA replicated as episomal, unit length genome and remained episomal after multiple passages. These data suggested the basis for an in vitro replication assay for evaluating the antiviral activity of potential chemotherapeutic agents directly on HPV. This model was used to investigate antiviral activities of current anti-HPV therapies such as 5-fluorouracil (5-FU) and alpha-interferon (alpha-IFN) and potential therapies such as sodium butyrate, 5-bromo-20-deoxyuridine (BrdU) and antisense oligonucleotides. HPV- 11 replication is significantly inhibited by BrdU and sodium butyrate; however 5-FU and alpha-IFN did not give consistent dose response results. Finally, ISIS 2105, a 20-mer phosphorothioate antisense oligonucleotide, which targets HPV-11 E2 gene product, showed potent antiviral activity in this assay with an IC50 of approximately 70 nM.

Antiviral Agents↗
PubMed1990

The role of dam methyltransferase in the control of DNA replication in E. coli.

The timing and control of initiation of DNA replication in E. coli was studied under conditions where the cellular level of dam methyltransferase was controlled by a temperature-inducible promoter. Flow cytometry was used to demonstrate that the synchrony of initiation at the several origins within each cell was critically dependent on the level of dam methyltransferase. Initiations were shown to be synchronous only in a narrow temperature range. The data are explained by a model where a newly replicated and therefore hemimethylated oriC is inert for reinitiation. Such a model may be applicable to eukaryotic cells, where classes of origins are initiated in synchrony and only once per cell cycle.

DNA Replication↗
PubMed2026

Replication stress links Geminin depletion to centrosome amplification.

The timing of DNA replication and centrosome duplication is tightly regulated with cell cycle progression to ensure the faithful duplication of the genome during cell division. Both DNA and centrosomes are licensed for replication in late telophase/early G1, replicated in S phase and segregated during mitosis; yet how defects in DNA replication licensing are coupled to centrosome homeostasis remains poorly understood. Here, we show that depletion of the replication licensing inhibitor Geminin in proliferating mouse embryonic fibroblasts induces robust centrosome amplification together with impaired primary cilium assembly. Rather than promoting whole-genome reduplication, knockdown of Geminin triggers a replication stress response, characterized by DNA damage accumulation throughout the cycle, and activation of an ATR-dependent DNA damage response. Mechanistically, Geminin depletion-induced replication stress activates the ATR-Chk1-Wee1 checkpoint axis prolonging G2 and leading to premature centriole disengagement and centrosome amplification. These findings identify replication stress as the signaling module that couples defective DNA replication licensing to centrosome amplification.

DNA damage↗
PubMed1986

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv.

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37 degrees C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13-18 times slower than that of E. coli.

Chloramphenicol↗
PubMed1987

A model for the spatio-temporal organization of DNA replication in mammalian cells.

The spatio-temporal organization of chromosomal DNA replication was analyzed using a model based on a "DNA unit" (or decondensation unit) hypothesis. The model is an extension of the fork movement theory of Huberman & Riggs (1968) and can account for a partially deterministic and partially stochastic order of DNA replication in chromosomes. It presumes that each chromosome is composed of DNA units that are arranged in sequence and that are replicated in parallel. A deterministic wave of chromatin decondensation propagates along the DNA unit continuously and progressively providing a field for the random activation of replication origin. Assignment of replication times to DNA compartments by a Monte Carlo method was programmed based on the model and the program was used to stimulate DNA synthesis rate curves that can be measured by the method of Dolbeare et al. (1983, 1985). The shape of the curve is shown to constrain possible parameter values of the model, which include the rate of fork movement, the fraction of chromatin that is decondensed at the start of S-phase, the initial number of origins activated, the rate at which new origins are activated, etc. The chromosomal organization that controls the molecular level of DNA replication is briefly reviewed and its relevance to the model is also discussed.

Algorithms↗
PubMed1994

A model for the correlation of mutation rate with GC content and the origin of GC-rich isochores.

Based on the biochemical kinetics of DNA replication and mutagenesis, including misincorporation and correction, a model has been developed for studying the relationships among the mutation rate (u), the G+C content of the sequence (f), and the G+C proportion in the nucleotide precursor pool (N). Also a measure for the next-nucleotide effect, called the maximum capacity of the next-nucleotide effect (MC), has been proposed. Under the normal physiological conditions of mammalian germ cells, our results indicate: (1) the equilibrium G+C content in a sequence is approximately equal to the G+C proportion in the nucleotide precursor pool, i.e., f approximately N, which is independent of the next-nucleotide effect; (2) an inverted-V-shaped distribution of mutation rates with respect to G+C contents is predicted, when the next-nucleotide effect is week, i.e., MC approximately 1; (3) the distribution becomes flatter (i.e., inverted-U-shaped) as MC increases, but the peak at 50% GC is still observed when MC < 2; and (4) the peak disappears when MC > 2.8, that is, when the next-nucleotide effect becomes strong. Our results suggest that changes in the relative concentrations of nucleotide precursors can cause variations among genes both in mutation rate and in G+C content and that compositional isochores (DNA segments with a homogeneous G+C content) can arise in a genome due to differences in replication times of DNA segments.

Base Composition↗
PubMed1995

c-Fos is involved in the cellular defence against the genotoxic effect of UV radiation.

The proto-oncogene c-fos encodes a nuclear protein that forms together with c-Jun or other members of the Jun family the transcription factor AP-1. The c-fos gene is inducible by UV radiation and other DNA damaging treatments which may indicate that it is required in defence against DNA damaging agents. To address this hypothesized function of c-Fos, we have compared the response of mouse fibroblasts deficient in c-Fos with the corresponding wild-type cells towards the genotoxicity of UV radiation. It is shown here that lack of c-Fos renders cells hypersensitive to the cytotoxic effect of UV light and gives rise to significant increases of UV-induced chromosomal mutations and DNA breakage. Cells lacking c-Fos were basically able to perform UV-induced repair replication, as measured by unscheduled DNA synthesis. However, with high doses of UV c-Fos deficient cells proved to be less efficient in repair synthesis than wild-type cells. Measurement of overall DNA synthesis after UV irradiation revealed that cells deficient in c-Fos are more inhibited in their recovery from the UV-induced block to replication. These data strongly suggest that c-Fos is involved in regulating the timing of DNA replication after UV irradiation by abolition of the UV-induced block to replication and thus appears to play a decisive role in the cellular defence against the genotoxic effects induced by UV radiation.

3T3 Cells↗
PubMed1994

Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos.

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

Cell Cycle↗
PubMed1996

cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication.

A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termed ROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM3 subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2-4 copies per haploid genome of closely related ROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels of ROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript. In situ hybridisation analysis in the root apex reveals that ROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley and Arabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.

Amino Acid Sequence↗