Search PubMed⌕ Search

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.

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

At least 469 records · Page 26Linked to original sources
PubMed1986

Post-meiotic DNA synthesis in nocodazole-blocked nuclei during conjugation of Tetrahymena thermophila. Induction of polyploidy in the micronucleus.

The formation of single, polyploid micronuclei was induced during conjugation of Tetrahymena thermophila with Nocodazole (ND) according to Kaczanowski et al. The increase in DNA content in these nuclei was measured cytophotometrically in conjugating pairs continuously exposed to the drug. ND-treated micronuclei ('restitution nuclei') undergo two complete rounds of DNA replication and enter the third at the time of pair separation. The DNA content of these micronuclei in late pairs was in the range 16-30C (mean 20C). This amount was similar to the sum of the DNA content of all post-meiotic products during normal conjugation at about the same stage. Thus the increase in DNA content in 'restitution' nuclei reflects some intrinsic ability of nuclei in pairing cells to replicate DNA independently of nuclear division.

Animals↗
PubMed1997

Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus.

DNA replication in the dimorphic bacterium Caulobacter crescentus is tightly linked to its developmental cell cycle. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell. To identify the signals responsible for the cell cycle control of DNA replication initiation, we have characterized a region of the C. crescentus chromosome containing genes that are all involved in DNA replication or recombination, including dnaN, recF, and gyrB. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. It is transcribed from three promoters; one is heat inducible, and the other two are induced at the transition from swarmer to stalked cell, coincident with the initiation of DNA replication. The single gyrB promoter is induced at the same time point in the cell cycle. These promoters, as well as those for several other genes encoding DNA replication proteins that are induced at the same time in the cell cycle, share two sequence motifs, suggesting that they represent a family whose transcription is coordinately regulated.

Amino Acid Sequence↗
PubMed2006

The temporal program of DNA replication: new insights into old questions.

During the last decades it has been shown that the replication timing program in metazoans is related to chromosome structure, the nuclear positioning and AT/GC content of chromosomal loci, their patterns of histone modifications, and their transcriptional regulation. Here, the current state of knowledge concerning these relationships is reviewed. An integrated view on structure-function relationships in the nucleus is provided and the determination and functional role of the replication timing program is discussed in this context. A corresponding comprehensive model is developed and a key aspect of this model is the suggestion that mammalian chromosomes are organized into stable units equivalent to replicon clusters. It is proposed that the nuclear positions of these units would depend on their histone modifications and determine the replication timing of the whole unit. It is furthermore predicted that replication timing is only indirectly linked to transcriptional regulation and contributes to the maintenance of gene expression patterns. These clear predictions, and the fact that the tools are at hand now to further test them, open an avenue towards solving the long standing problem on how replication timing is determined in metazoan cells.

Animals↗
PubMed2005

Human protein tau represses DNA replication in vitro.

Here, in the experiments of both PCR and real-time PCR, a repression of DNA amplification was observed in the presence of protein tau. Furthermore, a strong repression appeared when an in vitro DNA replication assay was performed at the physiological temperature (37 degrees C). The incorporation of dNTP was markedly decreased to approximately 12% of control by the presence of tau23 and to approximately 15% by tau40. In the competitive experiments, the PCR product could be restored when the competitor DNA was added, indicating that the association of tau with the template gave rise to the repression. However, tau did not repress the yield of RNA in transcription, suggesting that tau was replaced or ejected from the template by the elongating T7 RNA polymerase.

DNA↗
PubMed1984

Invariant temporal order of replication of the four actin gene loci during the naturally synchronous mitotic cycles of Physarum polycephalum.

The chronological sequence of replication for the four unlinked actin gene loci of Physarum has been established. Southern hybridization analysis of density-labeled, bromodeoxyuridine-substituted DNA isolated from defined periods of S phase demonstrates that three actin loci (ardB, ardC, ardD) are duplicated early, corresponding to the first 10% of the genome. The fourth locus (ardA) replicates later, between 80 and 100 min into S phase and after 75% of DNA synthesis is completed. Gene-dosage determinations, based on the quantitation of hybridization signals from DNAs isolated from various times during S phase, confirm the results obtained with bromodeoxyuridine-substituted DNA and increase the temporal resolution. The chronological order of replication in the macroplasmodium appears constant through two consecutive cell cycles and after prolonged growth in suspension culture. The precise chronology of DNA synthesis at the gene level extends to the coordinate replication of allele pairs.

Actins↗
PubMed1984

DNA replication of simian virus 40 chromatin in digitonin-treated and saponin-treated permeable cells.

Permeable cell systems have been developed by treatment of simian virus 40 (SV40)-infected cells with digitonin or saponin for studying in vitro DNA replication of chromatin. SV40 DNA replicated efficiently in these systems in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. Analysis of the time course of the replicated SV40 DNA I, II and replicating intermediates by gel electrophoresis and autoradiography indicated that SV40 DNA replicated completely in the form of nucleoprotein complexes (SV40 chromatin).

Animals↗
PubMed1990

Asynchronous replication of homologous loci on human active and inactive X chromosomes.

The two X chromosomes in mammalian females replicate asynchronously, the inactive later than the active one. Using BrdUrd-sensitive restriction and UV irradiation to identify newly synthesized DNA directly on Southern blots, and restriction fragment length differences to discriminate alleles on active and inactive human X chromosomes, we examined the replication of hypoxanthine phosphoribosyltransferase (HPRT) and clotting factor IX (F9) loci in clonal populations of mouse-human hybrids. We find that HPRT replicates at different times during the period of DNA synthesis (S phase), depending on its activity: It replicates in early S phase, when expressed (on the active X chromosome), and in late S phase when silent (on the inactive X chromosome). Furthermore, when reactivated, the derepressed locus is earlier replicating, supporting a relationship between replication and transcription. Neither F9 allele is expressed in these cells, and both replicate in the second half of S phase, (slightly earlier on active than on inactive X chromosome).

Alleles↗
PubMed1998

The S phase: beginning, middle, and end: a perspective.

Events in the S phase of the cell cycle have been investigated to a relatively limited extent in comparison with those in G1 and M phases. Four aspects of S are briefly discussed in this report: (1) the final biochemical step permitting initiation of DNA synthesis, (2) determination of replication timing of individual genes and its mechanism, (3) S phase processes that lead to the onset of M phase, and (4) resetting the S-phase machinery.

Animals↗
PubMed2006

Characterization of AcMNPV with a deletion of me53 gene.

The Autographa Californica multiple nucleopolyhedrovirus (AcMNPV) me53 gene, which was previously reported as one of the major early-transcribed genes, was deleted through homologous recombination from an AcMNPV genome propagated as a bacmid DNA in E. coli, generating a me53 gene knockout bacmid. Green fluorescent protein (GFP) expression analysis and supernatant passage assay revealed that the me53 knockout bacmid was unable to replicate in cell culture, while me53 repair bacmid, which was generated by reinsertion of the me53 gene into the mutant at a different locus (the gentamicin locus) with ET-recombination technique, resumed viral replication ability at wild-type levels, indicating that the defective phenotype of the me53 knockout virus was directly due to the deletion of the me53-ORF. Subsequent electron microscopy revealed that the me53 knockout bacmid failed to form nucleocapsid in the nuclei of the transfected cells, though viral infection seemed to be initiated. Meanwhile, real-time PCR analysis based on SYBR Green fluorescence indicated abolishment of the viral DNA replication by me53 gene inactivation. Thus, it is demonstrated for the first time that me53 knockout blocked viral DNA replication, nucleocapsid formation, and consequent BV and ODV production.

Animals↗
PubMed2000

Identification and molecular characterization of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus genomic region encoding the regulatory genes pkip, p47, lef-12, and gta.

Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV) is a baculovirus pathogenic to spruce budworm, the most damaging insect pest in Canadian forestry. CfMNPV is less virulent to its host insect and its replication cycle is slower than the baculovirus type species Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) but the basis of these characteristics is not known. We have now identified, localized, and determined the sequence of the region of CfMNPV carrying potentially important regulatory genes including p47, lef-12, gta, and pkip. DNA database searches revealed that this region of CfMNPV is most closely related to the homologous OpMNPV genes. Transcription analysis demonstrated that CfMNPV P47 is encoded by a 1.6-kb transcript, LEF-12 is encoded by a 2.6-kb transcript, and GTA is encoded by a 2.1-kb transcript. Transcripts for these genes were detectable at 6 h postinfection but all of them showed a burst in expression levels between 12 and 24 h postinfection corresponding to the time of initiation of CfMNPV DNA replication. A polyclonal antibody, raised against CfMNPV P47, detected a nuclear 43-kDa polypeptide from 12 to 72 h postinfection, demonstrating that the CfMNPV p47 gene product is first expressed at a time corresponding to the burst of transcriptional activity between the early and the late phases. Both AcMNPV and CfMNPV P47 translocate to the nucleus of infected cells.

Amino Acid Sequence↗
PubMed1995

Cell cycle checkpoints and DNA repair preserve the stability of the human genome.

Chemical carcinogenesis in the regenerating rat liver is cell-cycle-dependent. Proliferating hepatocytes were maximally susceptible to initiation by a single dose of benzo[a]pyrene diolepoxide I when at the G1/S border. Hepatocytes in early G1 or late S/G2/M were less susceptible and non-proliferating G0 hepatocytes were resistant to initiation. Radiation clastogenesis in proliferating human fibroblasts also is cell-cycle-dependent. Ultraviolet radiation (UV) induced maximal frequencies of chromosomal aberrations in synchronized cells that were at the G1/S border. Cells in early G1 or G2 were significantly less sensitive. For both initiation of chemical carcinogenesis and UV-clastogenesis, it appears that replication of damaged DNA is required and DNA repair before replication reduces cellular risk. If DNA repair is protective, cell cycle checkpoints which delay DNA replication and mitosis should augment this protective influence by providing more time for repair. The contribution of cell cycle checkpoint function to DNA repair during cell cycle-dependent clastogenesis was studied using ataxia telangiectasia (AT) fibroblasts. The AT cells displayed a defect in the coupling of DNA damage to checkpoints which control the G1/S and G2/M transitions and the rate of replicon initiation in S phase cells. UV-clastogenesis in AT cells was cell-cycle-dependent with irradiation at the G1/S boundary inducing 3-times more aberrations than treatment in G0 at the time of release into the cell cycle. Thus, DNA excision repair during the pre-replicative G1 phase was protective even in cells with defective checkpoint function. However, following irradiation at the G1/S border, AT cells displayed about 6-fold increased levels of UV-induced chromosome aberrations in comparison to normal human fibroblasts that were treated at this time. These observations indicate that secondary and tertiary DNA lesions that are produced during replication of UV-damaged DNA (replicative gaps and double-strand breaks) also depend on checkpoint function for repair. The replicon initiation and G2-delay checkpoints that operate after initiation of S phase appear to play a major role in protection against UV-clastogenesis.

Animals↗
PubMed1999

Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase.

Production of gutted, or helper-dependent, adenovirus vectors by current methods is inefficient. Typically, a plasmid form of the gutted genome is transfected with helper viral DNA into 293 cells; the resulting lysate is serially passaged to increase the titer of gutted virions. Inefficient production of gutted virus particles after cotransfection is likely due to suboptimal association of replication factors with the abnormal origins found in these plasmid substrates. To test this hypothesis, we explored whether gutted virus production would be facilitated by transfection into cells expressing various viral replication factors. We observed that C7 cells, coexpressing adenoviral DNA polymerase and preterminal protein, converted plasmid DNA into replicating virus approximately 50 times more efficiently than did 293 cells. This property of C7 cells can be used to greatly increase the efficiency of gutted virus production after cotransfection of gutted and helper viral DNA. These cells should also be useful for generation of recombinant adenovirus from any plasmid-based precursor.

Adenoviridae↗
PubMed1989

[Disruption of DNA replication in non-irradiated cells in basal cell nevus syndrome and the effect of ionizing radiation].

Analysis of DNA fiber autoradiograms from basal cell nevus syndrome (BCNS) skin fibroblasts has revealed for the first time a new defect in DNA replication earlier unknown in other chromosomal instability syndromes, that involves a significantly decreased rate of DNA-chain growth in unirradiated cells. Here we present evidence that the defect may be due to a marked reduction in number of simultaneously operating groups of replicons compared to that in normal cells, the rate of fork movement and the fusion of neighbouring units in the group remaining unchanged. Radioresistant DNA synthesis was observed in the BCNS cells. The exposure of cells derived from normal donor to gamma-rays at a dose of 5 Gy reduces the number of simultaneously operating groups of replicons to the level occurring in unirradiated BCNS cells, the rate of folk movement being unchanged in both cell types. However, the incidence of fusion between neighbouring units within the group is lower in the cells exposed to gamma-rays, due perhaps to a radiation-induced lesion in the group. Thus, ionizing radiation reduces the rate of DNA synthesis to the same level, however from different initial levels. Our data suggest that the phenomenon of radioresistant DNA synthesis may be explained by the presence of the initial defect in DNA replication in BCNS or any other chromosomal instability disorders.

Adult↗
PubMed2002

G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome.

Stretches of guanines can associate in vitro through Hoogsteen hydrogen bonding to form four-stranded structures. In the HIV-1 central DNA flap, generated by reverse transcriptase at the end of retrotranscription, both the two 99 nt-long overlapping (+) strands contain two adjacent tracts of guanines. This study demonstrates that oligonucleotides containing these G-clusters form highly stable G-quadruplexes of various structures in vitro, whose formation was controlled by an easy and reversible protocol using sodium hydroxide. Among these sequences, a G'2 hairpin dimer was the most stable structure adopted by the 5'-tail of the (+) downstream strand. Since the two (+) strands of the HIV-1 central DNA flap hold these G-clusters, and based on the properties of reverse branch migration in DNA flaps, constructions using HIV-1 sequences were assembled to mimic small DNA flaps where the G-clusters are neighbors. G-quartets were successfully probed in such flaps. They were induced by potassium and by a dibenzophenanthroline derivative already known to stabilize them. Such results suggest some function(s) for G-quartets associated with a DNA flap in the HIV-1 pre-integration steps, and argue for their transient formation during the processing of G-rich DNA flaps at the time of replication and/or repair.

DNA↗
PubMed1997

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.

Cell Cycle↗
PubMed1980

A novel mutant of bacteriophage T7 that is defective in early phage DNA synthesis.

A mutant of bacteriophage T7 is described which produces smaller plaques than the wild type and is defective in early phage DNA synthesis. The mutation is located in the Class II transcriptional region of the T7 genome together with all the other genes involved in phage DNA synthesis, but it could not be placed into any of the existing known T7 genes. DNA replication in strain R9 begins at the same time as for wild type although it proceeds very slowly until 15 minutes after infection, after which time DNA synthesis is apparently normal. It is concluded therefore that there are two types of DNA replication in phage T7, which differ with respect to their dependence on the mutant function. The change from one mode to the other is marked by the formation of folded, complex DNA inside the cell.

Chromosome Mapping↗
PubMed1989

Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29 DNA-protein p3 replication.

The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off.

Amino Acids↗
PubMed1985

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state.

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.

Animals↗