SEARCH · Search PubMed

Results for “DNA Replication Timing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources

PubMed2006

Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.

Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.

Animals↗

PubMed1986

Inhibition of DNA polymerase alpha by gossypol.

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase alpha (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase alpha with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase alpha does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase beta in a dose-dependent manner, but had no effect on DNA polymerase gamma. These results suggest that inhibition of DNA polymerase alpha may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.

DNA Polymerase I↗

PubMed2003

Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

Animals↗

PubMed1998

Cell reproduction and morphological changes in Mycoplasma capricolum.

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.

Bacterial Proteins↗

PubMed2003

Replication timing of the human genome.

We have developed a directly quantitative method utilizing genomic clone DNA microarrays to assess the replication timing of sequences during the S phase of the cell cycle. The genomic resolution of the replication timing measurements is limited only by the genomic clone size and density. We demonstrate the power of this approach by constructing a genome-wide map of replication timing in human lymphoblastoid cells using an array with clones spaced at 1 Mb intervals and a high-resolution replication timing map of 22q with an array utilizing overlapping sequencing tile path clones. We show a positive correlation, both genome-wide and at a high resolution, between replication timing and a range of genome parameters including GC content, gene density and transcriptional activity.

Base Composition↗

PubMed2004

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1.

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.

Aldehyde Dehydrogenase↗

PubMed1976

[Development of spontaneous mutations in somatic mamalian cells and DNA replication. II. Expression of gene mutations in cultured cells].

The dependence of spontaneous rate and phenotypic expression of 6-mercaptopurine resistance mutations of DNA replication synthesis was studied in cultured Chinese hamster cells. Spontaneous mutations arising with a constant rate per cell per time unit independently on DNA replication rate were shown to be expressed only in the course of subsequent cell divisions. The frequency of N-nitrosomethylurea induced mutations in cells with reduced and normal DNA replication rate is approximately the same. However, DNA replication synthesis is necessary for the phenotypic expression of both induced and spontaneous mutations. The causes of the phenotypic lag are discussed.

Animals↗

PubMed1999

Making CENs of mammalian artificial chromosomes.

Mammalian artificial chromosomes (MACs) hold the promise of providing autonomous vectors for gene therapy in dividing cells. They would not require insertion into the genome and could include sufficient genomic sequences that surround the therapeutic gene to ensure proper tissue-specific and temporal regulation. Several groups have reported successful formation of MACs in human cells using transfection strategies that included alpha satellite DNA, the primary DNA found at normal human centromeres. These results, although extremely encouraging, have limitations such as unpredictable chromosome formation and success thus far in only one transformed human cell line. Examination of other cells where alpha satellite DNA has integrated into ectopic chromosomal locations, as well as naturally occurring dicentric and neocentromere-containing cell lines, suggests that alpha satellite DNA may not be necessary or sufficient for centromere formation. Overall, these results suggest that epigenetic modifications of centromeric DNA are required for efficient centromere formation. Models for this centromere-specific epigenetic modification include a specialized chromatin structure and differential replication timing of centromeric DNA. Thus, further investigation of these centromere-specific epigenetic modifications may suggest strategies for increasing the efficiency of generating human artificial chromosomes for use as gene therapy vectors.

Animals↗

PubMed2002

Answering the Call: Coping with DNA Damage at the Most Inopportune Time.

DNA damage incurred during the process of chromosomal replication has a particularly high possibility of resulting in mutagenesis or lethality for the cell. The SOS response of Escherichia coli appears to be well adapted for this particular situation and involves the coordinated up-regulation of genes whose products center upon the tasks of maintaining the integrity of the replication fork when it encounters DNA damage, delaying the replication process (a DNA damage checkpoint), repairing the DNA lesions or allowing replication to occur over these DNA lesions, and then restoring processive replication before the SOS response itself is turned off. Recent advances in the fields of genomics and biochemistry has given a much more comprehensive picture of the timing and coordination of events which allow cells to deal with potentially lethal or mutagenic DNA lesions at the time of chromosomal replication.

Journal Article↗

PubMed1990

In vitro DNA replication by cytoplasmic extracts from cells infected with African swine fever virus.

A cell-free system that catalyzes DNA replication was prepared from cytoplasmic extracts of Vero cells infected with African swine fever virus (ASFV). The cells were permeabilized with lysolecithin and disrupted by mild mechanical action and the nuclei were removed by low-speed centrifugation. Extracts prepared from infected cells at the time of maximal DNA replication incorporated [alpha-32P]dTTP into acid-insoluble material that was sensitive to DNase and resistant to RNase. The reaction was inhibited by phosphonoacetic acid, an inhibitor of ASFV-specific DNA polymerase. Extracts from mock-infected cells had a negligible activity. Micrococcal nuclease-treated extracts were able to replicate added virion DNA or viral replicative DNA. An increase in the mass of DNA detected by ethidium bromide staining and by dot blot hybridization with ASFV DNA showed that the incorporation was due to true replication. Plasmid DNA was also replicated, which indicates that ASFV-specific DNA polymerase does not require a virus-specific origin of replication. The pattern of fragments generated by EcoRI digestion of the in vitro product was characteristic of viral replicative DNA. Hybridization with a recombinant plasmid containing a terminal fragment of ASFV DNA confirmed the presence of dimer terminal ASFV fragments presumably generated from concatemeric replicative intermediates.

African Swine Fever Virus↗

PubMed2002

Cmk2, a novel serine/threonine kinase in fission yeast.

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.

Amino Acid Sequence↗

PubMed1981

Segregation of chromosomes and DNA replication in intraspecific L X LS/BL hybrids.

The initial and terminal patterns of DNA replication in the chromosomes of the hybrids arising from a fusion of mouse lymphosarcoma LS/BL cells and L cells (HGPRT-) were studied. The replication of chromosomes in the hybrids compared to the parent cells was not modified by the alterations in generation time of the hybrids not by segregation of telocentric chromosomes. It can be assumed that there is a gross control system in LS/BL X L hybrids that is responsible for the patterns and timing of chromosome DNA replication and does not depend on interactions between genomes of the parent cells.

Animals↗

PubMed2002

Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract.

The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.

Animals↗

PubMed1978

DNA replication pattern and cell wall growth in Escherichia coli PAT 84.

An electron microscopic radioautographic study was made of tritiated thymidine incorporation into the genome of Escherichia coli PAT 84 and of tritiated meso-D,L-2,6-diaminopimelic acid (DAP) into the cell envelope. Pulse-labeled cells growing at 30 degrees C with a doubling time of 170 min were classified according to length by the method of agar filtration. Mathematical analysis of the length distribution led to the assumption of an exponential relation between length and time. A novel DNA replication pattern was found. Within the cell cycle DNA replication terminates at 70 min; then a gap follows of 64 min, after which DNA replication is initiated at 134 min. Thus, the C period is 106 min and the D period is 100 min. Cell constriction starts at 141 min and coincides with initiation of DNA replication. Detailed quantitative analysis of the [3H]thymidine grain frequency distribution allowed the distinction of three groups of cells. The first group incorporated no label, the second group an amount C, and the third group an amount 2 X C. The relative contribution of each group to a particular length class was determined. The data fitted very well into the DNA replication pattern. The same analysis was carried out on DAP pulse-labeled cells. Again, three groups of cells could be distinguished, and their relative contributions to each length class was determined. The group with the double amount of label was especially prominent at the end of the cell cycle. The emergence of this group might represent the acquisition of new lateral growth areas.

Cell Cycle↗

PubMed1978

Regulation of ribosomal RNA synthesis in Tetrahymena pyriformis.

Ribosomal RNA is synthesized at constant rate during most of the cell cycle in heat-shock synchronized populations of Tetrahymena pyriformis. Early in each macronuclear S-period the rate of synthesis increases abruptly, concomitant with replication of the genes coding for ribosomal RNA. The increase is prevented by inhibitors of DNA replication, added prior to the S-period. Similarly, in cultures synchronized by starvation/refeeding, inhibition of DNA replication, at the time when the rDNA is replicated, will prevent the normal increase in rate of RNA synthesis which follows refeeding. We conclude that inhibition of rDNA replication interferes with the synthesis of rRNA, and we suggest that with respect to rRNA synthesis a gene dosis effect is operating in fast-growing Tetrahymena cells.

Animals↗

PubMed1997

Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells.

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid fibroblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34(cdc2). These changes in mitotic control were evident within 5-10 population doublings after retroviral infection, indicating a direct effect of T expression. Cells acutely infected with the T-expressing retrovirus suffered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These findings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.

Antigens, Viral, Tumor↗

PubMed2004

A rapid in vitro polyomavirus DNA replication assay.

Traditionally, the Hirt extraction method, a multi-step, labor-intensive and time-consuming procedure, is employed to extract selectively low-molecular weight DNA for polyomavirus DNA replication analyses. DNA replication results obtained with this approach are often inconsistent between replicate samples. To increase the efficiency and reproducibility of the polyomavirus DNA replication assay, we compared the DNA quality and yield using Qiagen Spin Column technology and the Hirt extraction technique. CV-1 cells transfected with SV40 DNA were harvested at days 2, 4, and 6 post-transfection, and DNA was extracted using the Qiagen Spin Column and the Hirt extraction methods. Southern hybridization was performed using a (32)P-labeled linear full-length SV40 DNA probe. Viral DNA replication was quantitated using a BioRad phosphorimager, and results obtained with the two procedures were compared. Southern blot analysis revealed consistent and enhanced SV40 DNA recovery using the Qiagen Spin Column technology, and viral DNA replication over a 6-day period was reproducible among triplicate samples. In addition, Qiagen Spin Column technology reduced the time required to obtain good quality DNA for polyomavirus replication assays from 24 h to less than 3 h. Adoption of this extraction procedure will improve the determination of polyomavirus DNA replication activity, while reducing the investigator's exposure to and disposal of toxic organic compounds.

DNA, Viral↗

PubMed2000

The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation.

BACKGROUND: Sporulation of the fission yeast Schizosaccharomyces pombe is a cell differentiation process which accompanies meiosis. The spo6+ gene was identified as a sporulation-specific gene, whose transcription was regulated by the forkhead family transcription factor Mei4. RESULTS: spo6+ encodes a protein with sequence similarity to Saccharomyces cerevisiae Dbf4p, which is required for the initiation of DNA replication. However, doubling time and cell morphology of spo6 deletion mutants and spo6-cDNA over-expressing cells were indistinguishable from wild-type cells. Spliced mature mRNAs of spo6+ appeared when diploid cells committed to meiosis. Spo6p fused to green fluorescent protein (GFP) preferentially localized in a nucleus. Although spo6Delta diploids normally underwent premeiotic DNA replication and meiosis-I, approximately 80% of cells were blocked at the binucleate stage during meiosis and virtually no asci were formed. Anti-tubulin staining revealed that only 25% of the binucleate cells assembled spindle microtubules for meiosis-II. In a small number of tetranucleate cells, sister nuclei insufficiently separated and spindles were frequently fragmented. The meiosis-II arrest phenotype was exaggerated at low temperature and in the presence of caffeine. CONCLUSIONS: These results indicate that Spo6p is a novel Dbf4-related nuclear protein, which is expressed during meiosis and is indispensable for normal progression of meiosis-II and sporulation.

Amino Acid Motifs↗