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At least 1,549 records · Page 86Linked to original sources
PubMed1993

Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.

Adenosine Deaminase↗
PubMed1996

Mapping of polyomavirus DNA replicative intermediates by two-dimensional gel analysis using chemiluminescent detection.

Py DNA replicative intermediates (RIs) were mapped using a neutral/neutral two-dimensional (N/N 2D) technique by both chemiluminescent and radioactive detection. The nonradioactive method provides similar sensitivity to radioactive methods while possessing the advantages of stability of labeled probes, faster processing, multiple exposures, easy disposal and safety associated with nonradioactive detection. Application of this method to 2D gel analysis requires some technical modification to reduce background and save the substrate. The patterns of Py RIs with Afl II digestion suggest that bidirectional replication occurs during Py DNA synthesis in cell culture, but other unexpected structures of DNA replication are also seen.

Animals↗
PubMed2004

The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication.

The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.

Cell Cycle Proteins↗
PubMed1977

Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication.

Temperature-sensitive (ts) A 1S9 mouse L cells continue to synthesize double-stranded covalently closed mitochondrial (mt) DNA at a temperature (38.5 degrees C) which is nonpermissive for chromosomal DNA replication. The amount of mt DNA made appears to be quantitatively linked to nuclear DNA synthesis. Nuclear DNA replication proceeds normally for 6-8 h after the cells are shifted to 38.5 degrees C, and then declines to reach a minimum at 20-24 h. The level of mt DNA synthesis remains high during this period and decreases once the ts lesion has been established.

Animals↗
PubMed2005

Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase.

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase. Pulse-chase experiments demonstrate that the RS of early-S-phase are maintained when chased through S-phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid-S-phase with only a slight decrease in late-S-phase. 3-D reconstruction of serial sections reveals a network-like organization of the RS in early-S-phase and confirms that numerous smaller-sized replication foci comprise the larger RS characteristic of late-S-phase.

DNA Replication↗
PubMed1996

Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe.

Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Recent work has suggested that the mitotic form of cyclin-dependent kinase (Cdc2/cyclin B) has a role in preventing re-replication of the genome before mitosis, but the relevant targets of this inhibition are unknown. In this report we present evidence that the mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of Cdc18, a critical regulator of S-phase entry. We found that the ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits Cdc2 phosphorylation of histone H1 or a Cdc18 fusion protein by directly interacting with the Cdc2/cyclin B complex. Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of Cdc18 protein by a largely post-transcriptional mechanism. Overexpression of SIC1, an unrelated cyclin-dependent kinase inhibitor from budding yeast, causes a similar accumulation of Cdc18 and also leads to re-replication. Our data link a potent inhibitor of Cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggest that inhibition of Cdc18 by cyclin-dependent kinases has an important role in ensuring that the genome is duplicated precisely once each cell cycle.

CDC2 Protein Kinase↗
PubMed2004

Bending of adenovirus origin DNA by nuclear factor I as shown by scanning force microscopy is required for optimal DNA replication.

Nuclear factor I (NFI) is a transcription factor that binds to the adenovirus type 5 (Ad5) origin of replication and recruits the adenovirus DNA polymerase, thereby stimulating initiation of DNA replication in vitro. Using scanning force microscopy, we demonstrate that NFI induces a 60 degrees bend upon binding to the origin. The A/T-rich region preceding the core recognition sequence of NFI influences the DNA bend angle, since substitution of A/T base pairs by G/C base pairs severely decreases bending. Mutations in the A/T-rich region do not affect binding of NFI to DNA. However, mutations that reduce the protein-induced bend lead to a loss of NFI-stimulated replication, indicating that DNA bending is functionally important. In contrast, basal initiation or DNA binding of the polymerase is not impaired by these origin mutations. We conclude that binding of NFI to the Ad5 origin causes structural changes in DNA that are essential for the stimulatory function of NFI in replication. We propose that NFI-induced origin bending facilitates the assembly of a functional initiation complex.

Adenoviruses, Human↗
PubMed1977

Isolation of the viral DNA replication complex from adeno-associated virus type 1-infected cells.

The replication complex active in adeno-associated virus type 1 (AAV-1) DNA synthesis in vitro was solubilized, with a nonionic detergent, from the nuclei of human embryonic kidney cells coinfected with AAV-1 and an early temperative-sensitive mutant (ts125) of human adenovirus type 5 at the nonpermissive temperature (40.5 degrees C). The complex sedimented with a mean size of 23S and contained parental AAV-1 DNA. Most of the DNA synthesized with the AAV-1 DNA replication complex in vitro was AAV-1 DNA, as revealed by DNA-DNA hybridization and sedimentation in a neutral sucrose gradient. However, it sedimented in an alkaline sucrose gradient as molecules smaller than AAV-1 DNA (14.4S). The AAV-1 DNA replication complex was not formed in cells infected with AAV-1 alone.

Adenoviridae↗
PubMed1990

Topoisomerase I and II cleavage of adenovirus DNA in vivo: both topoisomerase activities appear to be required for adenovirus DNA replication.

Sites of topoisomerase I and II cleavage across large portions of the adenovirus type 5 genome were mapped by using the drugs camptothecin and VM26, respectively. These drugs prolong the half-lives of the covalent DNA-protein intermediates in which the DNA is transiently cleaved, and so treatment with protein denaturants after exposure to the drugs leads to DNA strand scission at the site of topoisomerase cleavage. Strong topoisomerase II cleavage sites occurred in clusters throughout the regions examined, including both transcribed regions and transcriptional control regions. The efficiency of topoisomerase II cleavage increased as the rate of adenovirus DNA replication increased and then decreased with the decreasing rate of replication late in the infection cycle. The increase was not dependent on expression of the E1A gene, whose products activate transcription of the early viral genes. Positions of topoisomerase II cleavage sites did not vary during the infection. Topoisomerase I cleavage sites were also found throughout the examined regions, with the strongest sites occurring near the ends of the transcription units. Topoisomerase I cleavage in the E1 region occurred much more frequently than topoisomerase II cleavage, was not dependent on E1A gene expression, and remained at a similar level from the early viral phase into the late viral phase. Treatment of infected cells with either drug prevented efficient replication of adenovirus DNA. Inhibition of topoisomerase I activity led to an immediate cessation of adenovirus DNA replication, while inhibition of topoisomerase II blocked replication only after completion of approximately one additional round.

Adenovirus Early Proteins↗
PubMed1998

Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication.

In the mouse, transcriptional permissiveness is established in the fertilized egg prior to the activation of zygotic genes at the 2-cell stage. Therefore, gene inactivity initiated at the end of gametogenesis results from a complex process, involving more than an inhibition of the basal transcriptional apparatus. We have examined the ability of the first intron (I1) of the human hypoxanthine phosphoribosyl transferase gene, which functions as an enhancer in embryonic stem cells, to activate a reporter gene when placed proximally to or at a distance from the HSV-tk promoter, or when integrated into the mouse genome as part of a stable transgene. In microinjected embryos, I1 functions as an enhancer sequence; however, its competence for long-range activation appears only after the late 1-cell stage and depends on the first DNA replication. Moreover, activation of microinjected transgenes from proximal enhancers occurs in the late 2-cell embryo and in the male pronucleus of 1-cell embryos blocked for DNA replication; whereas, for integrated transgenes, proximal enhancer activity is subject to position effects in the 2-cell embryo and first occurs at the 2- or 4-cell stage, but only after completion of DNA replication. Therefore, the absence of long-range activation and a non-permissive genomic state (the relief of which both depend on DNA replication), together with an inactive transcriptional apparatus, appear to converge to prevent any gene activity in the 1-cell embryo. We propose that the embryo exploits the process of DNA replication to relieve the transcriptionally repressive state that was initially established to fulfil two purposes: (1) to arrest maternal gene expression in the maturing oocyte and (2) to protect the unicellular egg and 1-cell embryo from premature differentiation. Reactivation of gene expression by DNA replication would therefore serve to coordinate cell proliferation and differentiation in the preimplantation embryo.

Animals↗
PubMed1995

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

DNA Repair↗
PubMed2001

Chromatin remodeling and initiation of DNA replication.

While much has been learned in recent years about the process of chromatin remodeling and its role in activation of transcription, relatively little has been reported on the role of chromatin remodeling in DNA replication. However, it is well established that transcription factors and chromatin structure play an important role in replication origin usage. Recent work has begun to indicate that chromatin remodeling factors are likely to play an important role in the regulation of replication origin usage. The results to date are most consistent with the role for chromatin remodeling factors in DNA replication as being indirect, and very similar to their role in transcription. The current evidence suggests that transcription factors bind to auxiliary sequences adjacent to replication origins and recruit chromatin remodeling factors to create either nucleosome-free regions or regions of specifically spaced nucleosomes. This results in activation of the nearby origin, presumably by making the origin region more accessible to replication factors. Until recently, there has been very little evidence of direct interactions between chromatin remodeling factors and the DNA replication machinery. Recent studies have provided data indicating that direct interactions may exist between chromatin remodeling factors and two cellular replication factors, the Origin Recognition Complex and Proliferating Cell Nuclear Antigen. However, since these replication factors are also involved in other nuclear processes, such as transcriptional silencing and DNA repair, respectively, further study is necessary to establish whether these direct interactions are also important for DNA replication.

Animals↗
PubMed1993

Polyoma virus early-late switch: regulation of late RNA accumulation by DNA replication.

Early in infection of permissive mouse cells, messages from the early region of the polyoma virus genome accumulate preferentially over those from the late region. After initiation of DNA replication, the balance between early and late gene expression is reversed in favor of the late products. In previous work from our laboratory, we showed that viral early proteins do not activate the polyoma late promoter in the absence of DNA replication. Here we show that activation of the late genes in replication-incompetent viral genomes can occur if actively replicating genomes are present in the same cell. A low level of DNA replication, however, is insufficient to induce the early-late switch. Furthermore, replication-competent genomes that fail to accumulate late RNA molecules are defective in the transactivation of replication-incompetent genomes. We suggest that titration of an unknown diffusible factor(s) after DNA replication relieves the block to late RNA accumulation seen in the early phase, with most of this titration being attributable to late-strand RNA molecules themselves.

3T3 Cells↗
PubMed1998

Specificity of multiple homologous genomic regions in Spodoptera exigua nucleopolyhedrovirus DNA replication.

The region upstream of the Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) ubiquitin gene contains four near-identical 68-bp-long palindromic repeats. This region, named Sehr6 and located at map unit (m.u.) 88 of the SeMNPV genome on pSeEcoRI-2.2, showed structural homology to previously identified homologous regions (hrs) in a number of other baculoviruses. Hrs function as enhancers of transcription and as putative origins (oris) of baculovirus DNA replication. Five additional hrs (Sehr1-Sehr5) were identified on the SeMNPV genome by Southern blot hybridization with an 18-bp-long oligonucleotide complementary to a sequence conserved within the arms of the four palindromic repeats of Sehr6. Sehr1-Sehr6 were dispersed on the SeMNPV genome at m.u. 8.0, 30.0, 38.5, 51.0, 77.0 and 88.0, respectively. Sequence analysis of these hrs confirmed the presence of palindromic repeats, highly similar to those found in pSeEcoRI-2.2. The number of palindromes varied from one (Sehr4) to nine (Sehr1) per hr. The Sehrs are all present in non-coding regions of the SeMNPV genome and also contain multiple putative transcription recognition sequences. Plasmids containing either of the Sehrs replicated in an SeMNPV-dependent DNA replication assay. The Sehrs were unable to replicate in an AcMNPV-dependent DNA replication assay. This was in contrast to the previously observed SeMNPV non-hr type ori, which replicated in the presence of both AcMNPV and SeMNPV. These data suggest that the replication of SeMNPV and the role of hrs in this process is highly specific.

Animals↗
PubMed1990

[The role of protein binding with single-stranded DNA (SSB-protein) in DNA replication in Ehrlich ascites carcinoma cells].

Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.

Animals↗
PubMed1992

Inhibition of bovine papillomavirus plasmid DNA replication by adeno-associated virus.

The helper-dependent human parvovirus adeno-associated virus type 2 (AAV) inhibits both the oncogenic transforming abilities and the DNA replication of its helper viruses, adenovirus (Ad), and herpes simplex virus (HSV). As AAV-2 also inhibits the transforming ability of bovine papillomavirus type 1 (BPV), AAV-2 was assayed for its ability to inhibit BPV plasmid DNA replication. Here we find that the AAV-2 Rep78 gene is able to trans-inhibit BPV plasmid DNA replication and that the AAV-2 terminal repeats (TR) are also cis-required for the full inhibitory effect of AAV-2. When both the AAV-2 Rep78 open reading frame and TRs are present the inhibition of BPV plasmid DNA replication is very strong.

Animals↗
PubMed1996

An Autographa californica nucleopolyhedrovirus lef-2 mutant: consequences for DNA replication and very late gene expression.

In order to define factors involved in very late Autographa californica nucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1.lacZ) by 5'-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of the EcoRI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and in lef-2. Further mapping experiments demonstrated that only replacement of the point mutation in lef-2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that the lef-2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in the lef-2 gene of VLD1 did not have an effect on DNA replication. It is proposed that lef-2 may play a dual role, both in DNA replication and very late gene expression.

Amino Acid Sequence↗
PubMed2002

Involvement of DNA polymerase beta in DNA replication and mutagenic consequences.

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.

Animals↗