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At least 1,351 records · Page 75Linked to original sources
PubMed2002

A system for small-molecule control of conditionally replication-competent adenoviral vectors.

Replication-competent adenoviral vectors are potentially far more efficient than replication-defective vectors. However, for reasons of safety, there is a need to restrict viral replication both spatially, by limiting replication to certain cell types, and temporally. To control replication temporally, we have developed a system, based on the small-molecule dimerizer rapamycin, for regulating the replication of adenoviral vectors. In this system, one adenoviral vector, AdC4, expresses transcription factors whose activity is regulated by the non-immunosuppressive rapamycin analog AP21967. A second vector, Ad(Z12-I-E1aE1b19k), contains E1 genes placed downstream of binding sites for the regulated transcription factor. Co-infection of several cell lines by the vector pair leads to dimerizer-dependent E1 expression and an increase in viral replication, as shown by Southern blots and replication assays. Furthermore, expression of a reporter gene from a replication-defective vector, Ad-GM-CSF, can be augmented by up to 18-fold by co-infection with the pair of conditionally replicating vectors in the presence of dimerizer. Similar results are obtained when the vectors are directly injected into subcutaneous HT1080 xenograft tumors in nude mice. We believe that vectors based on this principle will be a useful additional tool to enhance efficiency and safety of gene delivery for anti-cancer therapy.

Adenoviridae↗
PubMed1993

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis.

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.

Cell-Free System↗
PubMed1989

How does inactivation change timing of replication in the human X chromosome?

The kinetics of replication of the inactive (late replicating) X chromosome (LRX) were studied in karyotypically normal lymphocytes and human amniotic fluid cells. Both cell types were successively pulse labeled with 1-h or 1/2-h thymidine pulses in an otherwise BrdU-substituted S phase after partial synchronization of the cultures at G1/S. For the first time with this technique, the entire sequence of replication was analyzed for the LRX from the beginning to the end of the S phase, with special reference to mid S (R-band to G-band transition replication). The inactive X is the last chromosome of the metaphase to start replication, with a delay of 1 or 2 h, after which time a thymidine pulse results in R-type patterns. In mid S, the inactive X is the first chromosome to switch to G-type replication (without overlapping of both types and without any detectable replication pause). Until the end of S, a thymidine pulse results in G-type patterns. To rule out artifacts that might arise by the synchronization of cultures in these experiments, controls were carried out with BrdU pulses and the BrdU antibody technique without synchronization. In the course of replication, no fundamental difference was seen between the two different cell types examined. In contrast to studies using continuous labeling, this study did not reveal an interindividual difference of replication kinetics in the LRXs of the seven individuals studied; thus it is concluded that the inactive X chromosome shows only one characteristic course of replication.

Amniotic Fluid↗
PubMed1993

The replication behavior of Saccharomyces cerevisiae DNA in human cells.

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.

Cell Line, Transformed↗
PubMed1984

Repair and replication of DNA in rat brain and liver during foetal and post-natal development, in relation to nitroso-alkyl-urea induced carcinogenesis.

The susceptibility of rat brain to induction of cancer by N-nitroso-N-ethyl-urea (NEU) increases dramatically from a very low level in the 12 day foetus to a maximum at the time of birth, and then decreases with age. Liver tumors are rarely induced by a single treatment with NEU at any age. If induction of cancer by nitroso-alkyl-ureas depends on replication of DNA containing the mispairing base O6-alkylguanine, susceptibility would reflect the balance between the protective effect of removal of the base by repair mechanisms and the potentiating effect of cell replication. The capacity of tissues to remove O6-alkyl-guanine from DNA depends on the amount of alkyl acceptor protein (AAP) present. To study the concept that carcinogenesis results from replication of alkylated DNA, the AAP contents and relative rates of DNA replication were studied in brain and liver of rats at various stages of development, from the 12 day foetus to the 48 week old rat. Replication in liver was approximately ten times higher than in intra-cranial contents at each stage of development studies. The AAP content was higher in the 12 day foetal brain than later, decreasing to low levels as susceptibility to NEU increased until the time of birth, and then remaining low in the adult. The peak sensitivity of brain therefore corresponds to the time at which AAP content is low and rate of DNA replication is high. With liver, AAP levels are low in the foetus, although higher than in brain, and increase after birth. The higher level of AAP in the foetal liver compared with that of brain is possibly sufficient to explain why cancer is not induced in liver in spite of the high rate of DNA replication. The results are consistent with the concept that replication of alkylated DNA is an essential event in initiation. There may be no quantitative relationship between replication and repair and susceptibility to cancer on comparison of different tissues, owing to the fact that, at the cellular level, cancer is a rare event. The amount of mispairing at replication necessary to bring it about may depend on the detailed organisation of the genome, and hence on cell type.

Age Factors↗
PubMed1992

Replication of the heterogeneous heterochromatin of the sex chromosomes of Microtus cabrerae.

We used bromosubstitution to investigate the mode of replication of different types of heterochromatin located in the sex chromosomes of Microtus cabrerae. Our results clearly show that, although the heterochromatin is late replicating, the replication timing of different types of constitutive heterochromatin is related to their banding properties: R-type heterochromatin replicates before G-type heterochromatin, and this replication is asynchronous in both sex chromosomes. Furthermore, the late replication behaviour of the inactive X may spread to its constitutive heterochromatin. In some cells, a region of the constitutive heterochromatin of the late replicating X spontaneously switches to early replication, which may be related to transcriptional activity. Replication behaviour of the constitutive heterochromatin in the Y chromosome is similar to that of the late replicating X.

Animals↗
PubMed2000

Immunoelectron microscopy of PCNA as an efficient marker for studying replication times and sites during pollen development.

Here we report for the first time the ultrastructural localization of DNA replication sites in the nucleus of plant cells and the timing of replication through the pollen developmental programme by proliferating cell nuclear antigen (PCNA) immunogold labelling. Replication sites were identified by labelling with anti-PCNA antibodies in fibrils of the interchromatin region close to the condensed chromatin, defining a perichromatin subdomain in the interchromatin space where DNA replication takes place. The same nuclear structures are decorated by anti-BrdU (5-bromo-2'-deoxyuridine) immunogold after short pulses of BrdU labelling. Double immunogold labelling for PCNA and DNA show colocalization on these perichromatin structures. PCNA immunoelectron microscopy also allows correlation of replicative activity with the dynamics of chromatin condensation. DNA replication was also monitored at different phases during pollen development by PCNA immunoelectron microscopy, revealing two peaks of DNA synthesis, at the beginning (early tetrad), and the end (late vacuolate), of microspore interphase. High-resolution autoradiography after [3H]thymidine incorporation also showed high replicative activity at the same two periods of microspore interphase. In the bicellular pollen grain, PCNA immunogold labelling revealed that DNA replication in the generative cell starts at an intermediate stage of pollen maturation, whereas the vegetative nucleus does not replicate and is arrested in G1. The use of anti-PCNA antibodies at the ultrastructural level is an easier, faster and more feasible method than the detection of in vivo-incorporated nucleotides, especially in plant systems with long cell cycles. PCNA immunogold labelling is, therefore, proposed as an efficient marker for mapping the sites and timing of replication at the electron microscopy level.

Autoradiography↗
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↗
PubMedDate not supplied

FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes.

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with FMR1 and X-chromosome alpha-satellite probes to interphase cells of the various genotypes: the alpha-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication associated with X-inactivation.

Adolescent↗
PubMed2005

A genomic view of eukaryotic DNA replication.

Recent advances in DNA microarray technology have enabled eukaryotic replication to be studied at whole-chromosome and genome-wide levels. These studies have provided new insights into the mechanisms that influence origin selection and the temporally co-ordinated activation of replication initiation from these sites. Here we describe multiple microarray-based approaches that have been used to study DNA replication in both S. cerevisiae and higher eukaryotes. We have also compiled the data from the yeast microarray-based replication studies to generate a comprehensive list of origins that has been verified in three independent studies. The comprehensive nature of the microarray-based studies has revealed clear connections between chromosome organization and the pattern of replication. For example, in yeast, the centromeric proximal sequences are consistently early replicating and telomeric regions are consistently late replicating. The metazoan studies reveal a recurring theme of gene-dense transcriptionally active regions of the genome replicating before gene-sparse regions. In addition to the insights they have provided already, microarray-based replication assays combined with genetic analysis will provide a powerful new approach to define the mechanisms that regulate replication origin function.

Animals↗
PubMed1983

Aberrant replication of chromosomal DNA segments in CHO cells as a manifestation of a DNA repair system.

We have previously demonstrated that transient blocking of DNA synthesis with drugs such as 1-beta-D-arabinofuranosylcytosine results in some segments of the chromosomal DNA being replicated more than once in a single cell cycle. One explanation for this phenomenon might be that it is due to a perturbation of the cellular mechanism which normally controls the process of initiating replication of the chromosomal DNA and which ensures that each DNA segment is normally replicated once only in each cell cycle. To examine an alternative explanation, we have used the Chinese hamster ovary CHO-K1 cell line to test whether UV irradiation induces aberrant double replication of chromosomal DNA segments. We present data which show that UV irradiation induces a linear increase (regression coefficient, 0.996) in aberrant reinitiation of DNA replication in DNA segments replicated earlier in the same cell cycle. This was shown by DNA strands labelled with bromodeoxyuridine (BrdU) being synthesized off 3H-labelled template strands which were themselves synthesized during a [3H]thymidine pulse shortly before the UV irradiation. We suggest that blocked replication forks trigger an emergency response to unreplicated DNA segments in which abnormal origins of replication are used to circumvent this damage. This results in abnormal patterns of DNA replication, and it occurs whether DNA replication forks are blocked metabolically (such as with an inhibitor of DNA polymerase) or physically (as in the case of pyrimidine dimers).

Animals↗
PubMed1996

Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells.

Peroxynitrite is a powerful oxidant formed through reaction of nitric oxide with superoxide. Because activated macrophages can produce both nitric oxide and superoxide, it has been proposed that peroxynitrite may contribute to cytotoxicity and increased cancer risks associated with the inflammatory response during chronic infections. We therefore investigated mutagenicity of peroxynitrite in the supF gene of the pSP189 shuttle vector as a mutation target. The plasmid was exposed to 2.5 mM peroxynitrite in vitro, then replicated in Eschericia coli MBL50 and in human AD293 cells. Mutation frequency increased 21-fold in pSP189 replicated in E. coli and 9-fold in plasmid replicated in human cells. Mutations were clustered within the 5' region of the supF gene in plasmids replicated in bacteria. The hot spots were located at positions 108, 113, 116, 124, 126 and 141; more than 25% of all mutations occurred at position 124. Following replication in human cells, mutations were more widely distributed over the gene, with hot spots at positions 113, 124, 133, 156 and 164; 15% occurred at position 124. In both systems, the majority of mutations occurred at G:C base pairs, predominantly involving G:C-->T:A transversions (65% when replication was in bacteria and 63% when in human cells). G:C-->C:G transversions were observed at lower frequency (28% in MBL50 and 11% in AD293 cells), and 11% of mutations found in vectors replicated in AD293 cells were G:C-->A:T transitions. A greater number of large deletions, insertions, tandem and multiple mutations occurred in plasmid replicated in AD293 cells. Differences in mutation spectra following replication in the two systems may be attributable to differences in recognition and repair of the lesions and/or properties of the replication apparatus.

Base Sequence↗
PubMed1990

Human cytomegalovirus major immediate early gene product can induce SV40 DNA replication in human embryonic lung cells.

Previously we had reported that human cytomegalovirus (HCMV) induced replication of plasmids containing the SV40 origin of replication in human fibroblasts that were nonpermissive for SV40 and permissive for HCMV DNA replication. The amplification of SV40 origin-containing plasmids was dependent upon the HCMV-induced expression of T-antigen RNA. From previous studies it was determined that cotransfection of cosmids, containing HCMV genomic DNA, could stimulate SV40 DNA replication and T-antigen production. This indicated that the gene products of HCMV responsible for inducing SV40 DNA replication could be determined. In this study we report that the cotransfection of the major IE gene of HCMV alone was sufficient to facilitate the replication of the SV40 origin-containing plasmid. The HCMV IE1 gene product (i) increased expression of T-antigen RNA and protein and (ii) induced SV40 plasmid DNA replication in a T-antigen-dependent manner. The SV40 replication event was not due only to the expression of T-antigen. When the gene coding for T-antigen was placed under control of the Rous sarcoma viral promoter so that T-antigen expression in HEL cells was constitutive, it was not sufficient to replicate the SV40 plasmid in the absence of the HCMV IE1 protein. Therefore, the major IE gene of HCMV was capable of increasing the expression of T-antigen RNA and facilitating the replication of the SV40 origin. We are currently investigating the mechanism responsible for these observations.

Animals↗
PubMed1983

ARS replication during the yeast S phase.

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.

DNA Replication↗
PubMed1983

Tumor promoter TPA increases initiation of replication on DNA injected into xenopus eggs.

The effect of the tumor promoter TPA on the control of DNA replication was assayed by following the regulated replication of DNA microinjected into eggs of the frog Xenopus laevis. TPA increases the amount of replication of injected DNA. Both initiation of replication on parental DNA molecules and reinitiation on previously replicated molecules are stimulated. Interaction with the external membrane appears necessary since injections of high concentrations of TPA into the egg are ineffective, whereas nM concentrations are active in the external medium. Related molecules that lack tumor promoting activity do not affect DNA replication. The effect of TPA on DNA replication was detectable only after the first cell cycle, and TPA cannot induce replication in oocytes, the quiescent stage which precedes the egg. When protein synthesis is inhibited TPA still increases initiation of replication, but does not allow detectable reinitiation cycles. The results suggest that interaction of TPA with the cell membrane is sufficient to increase the efficiency of replication initiation by a mechanism that does not require illegitimate reinitiation within a single cell cycle.

Animals↗
PubMed1988

Mechanism of plasmid pT181 DNA replication.

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. This nick presumably serves as the start-site of pT181 replication by extension synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. In vitro replication of a recombinant plasmid carrying two pT181 origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading-strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two PT181 origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.

Bacterial Proteins↗
PubMed2004

Checkpoint responses to replication fork barriers.

The fidelity of DNA replication is of paramount importance to the maintenance of genome integrity. When an active replication fork is perturbed, multiple cellular pathways are recruited to stabilize the replication apparatus and to help to bypass or correct the causative problem. However, if the problem is not corrected, the fork may collapse, exposing free DNA ends to potentially inappropriate processing. In prokaryotes, replication fork collapse promotes the activity of recombination proteins to restore a replication fork. Recent work has demonstrated that recombination is also intimately linked to replication in eukaryotic cells, and that recombination proteins are recruited to collapsed, but not stalled, replication forks. In this review we discuss the different types of potential replication fork barriers (RFB) and how these distinct RFBs can result in different DNA structures at the stalled replication fork. The DNA structure checkpoints which act within S phase respond to different RFBs in different ways and we thus discuss the processes that are controlled by the DNA replication checkpoints, paying particular attention to the function of the intra-S phase checkpoint that stabilises the stalled fork.

DNA Damage↗
PubMedDate not supplied

Replicative enzymes, DNA polymerase alpha (pol alpha), and in vitro ageing.

Normal cells in culture are used to investigate the underlying mechanisms of DNA synthesis because they retain regulatory characteristics of the in vivo replication machinery. During the last few years new studies have identified a number of genetic changes that occur during in vitro ageing, providing insight into the progressive decline in biological function that occurs during ageing. Maintaining genomic integrity in eukaryotic organisms requires precisely coordinated replication of the genome during mitosis, which is the most fundamental aspect of living cells. To achieve this coordinated replication, eukaryotic cells employ an ordered series of steps to form several key protein assemblies at origins of replication. Major progress has recently been made in identifying the enzymes, and other proteins, of DNA replication that are recruited to origin sites and the order in which they are recruited during the process of replication. More than 20 proteins, including DNA polymerases, have been identified as essential components that must be preassembled at replication origins for the initiation of DNA synthesis. Of the polymerases, DNA polymerase alpha-primase (pol alpha) is of particular importance since its function is fundamental to understanding the initiation mechanism of eukaryotic DNA replication. DNA must be replicated with high fidelity to ensure the accurate transfer of genetic information to progeny cells, and decreases in DNA pol alpha activity and fidelity, which are coordinated with cell cycle progression, have been shown to be important facets of a probable intrinsic cause of genetic alterations during in vitro ageing. This has led to the proposal that pol alpha activity and function is one of the crucial determinants in ageing. In this review we summarize the current state of knowledge of DNA pol alpha function in the regulation of DNA replication and focus in particular on its interactive tasks with other proteins during in vitro ageing.

Cell Cycle↗