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At least 847 records · Page 47Linked to original sources
PubMed1998

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.

Bacterial Proteins↗
PubMedDate not supplied

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA.

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).

DNA Replication↗
PubMed1997

Roles of replication protein-A subunits 2 and 3 in DNA replication fork movement in Saccharomyces cerevisiae.

Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants. Thus, replication fork movement in vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode.

Cell Cycle↗
PubMed1998

Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo.

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.

Adenine↗
PubMed1990

The roles of DNA polymerases alpha and delta in DNA replication.

The identities and precise roles of the DNA polymerase(s) involved in mammalian cell DNA replication are uncertain. Circumstantial evidence suggests that DNA polymerase alpha and at least one form of DNA polymerase delta, that which is stimulated by Proliferating Cell Nuclear Antigen, catalyze mammalian cell replicative DNA synthesis. Further, the in vitro properties of polymerases alpha and delta suggest a model for their coordinate action at the replication fork. The present paper summarizes the current status of DNA polymerases alpha and delta in DNA replication, and describes newly available approaches to the study of those enzymes.

Animals↗
PubMed1974

Adenovirus DNA replication. I. Requirement for protein synthesis and isolation of nuclear membrane fractions containing newly synthesized viral DNA and proteins.

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [(3)H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [(3)H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.

Adenoviridae↗
PubMed1996

Cdc6 and DNA replication: limited to humble origins.

The budding yeast Cdc6 protein is important for regulating DNA replication initiation. Cdc6p acts at replication origins, and cdc6-1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. Overexpression of the related Cdc18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. A recent paper further defines the role of Cdc6p in DNA replication. Cdc6p only promotes DNA replication between the end of mitosis and late G1, and although the Cdc6 protein is highly unstable, neither degradation nor nuclear localization is critical for limiting DNA replication to this interval.

Cell Cycle↗
PubMed2001

Mrc1 transduces signals of DNA replication stress to activate Rad53.

Cells experiencing DNA replication stress activate a response pathway that delays entry into mitosis and promotes DNA repair and completion of DNA replication. The protein kinases ScRad53 and SpCds1 (in baker's and fission yeast, respectively) are central to this pathway. We describe a conserved protein Mrc1, mediator of the replication checkpoint, required for activation of ScRad53 and SpCds1 during replication stress. mrc1 mutants are sensitive to hydroxyurea and have a checkpoint defect similar to rad53 and cds1 mutants. Mrc1 may be the replicative counterpart of Rad9 and Crb2, which are required for activating ScRad53 and Chk1 in response to DNA damage.

Amino Acid Sequence↗
PubMed2002

DNA replication and nuclear architecture.

The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.

Animals↗
PubMed1986

Chromatin assembly during SV40 DNA replication in vitro.

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.

Antigens, Polyomavirus Transforming↗
PubMed1993

MCB elements and the regulation of DNA replication genes in yeast.

In eukaryotic organisms, genes involved in DNA replication are often subject to some form of cell cycle control. In the yeast Saccharomyces cerevisiae, most of the DNA replication genes that have been characterized to date are regulated at the transcriptional level during G1 to S phase transition. A cis-acting element termed the MluI cell cycle box (or MCB) conveys this pattern of regulation and is common among more than 20 genes involved in DNA synthesis and repair. Recent findings indicate that the MCB element is well conserved among fungi and may play a role in controlling entry into the cell division cycle. It is evident from studies in higher systems, however, that transcriptional regulation is not the only form of control that governs the cell-cycle-dependent expression of DNA replication genes. Moreover, it is unclear why this general pattern of regulation exists for so many of these genes in various eukaryotic systems. This review summarizes recent studies of the MCB element in yeast and briefly discusses the purpose of regulating DNA replication genes in the eukaryotic cell cycle.

Bacterial Proteins↗
PubMed2001

In vivo association of Ku with mammalian origins of DNA replication.

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.

Animals↗
PubMedDate not supplied

The effect of Ni(II) on DNA replication.

The cellular regulation of DNA replication is governed in part by the availability of essential metal ions. A continuous supply of Mg(II) ions is necessary for the efficient and faithful replication of parental strands during S-phase as well as during the repair of DNA damage. A metal ion such as Ni(II) may interfere with the replication process by binding to sites on proteins at which essential ions normally bind. Binding at these sites by a toxic metal ion may produce inappropriate responses from the replication proteins and thus alter the normal balance in one or more of the microsteps comprising DNA synthesis. We have studied the effect of Ni(II) on DNA replication in a reconstituted in vitro system using a HeLa cell extract as a source of polymerase activity on a template of activated calf thymus DNA. Ni(II) has an initial stimulatory effect that is followed by an overall inhibition of the incorporation of DNA precursors. These results suggest that Ni(II), similar to Mg(II) may have more than one binding site, but that the binding of Ni(II) to replication proteins may significantly alter the timing of events in DNA synthesis.

Animals↗
PubMedDate not supplied

[Possibility of participation of proteins binding single-stranded DNA in eukaryotic DNA replication].

Main properties of the single-stranded DNA-binding proteins (SSB-proteins) (type of the protein UP 1 from the calf thymus) have been analyzed. The combination of some properties allows one to say about the strong specificity of the term "SSB-proteins type UP 1" and their differences from the HMG-proteins and other DNA-binding proteins and enzymes. Basing on the data from literature and the author's data the hypothesis was made that eukaryotic SSB-proteins may be involved in DNA replication, though they have tight relationship with the proteins of heterogeneous nuclear ribonucleoprotein particles. The following fact evidence for the involvement of SSB-proteins in DNA replication: 1) preferential affinity to the single-strand DNA in comparison with the single-strand RNA; 2) the contact between DNA SSB-proteins in chromatin; 3) strong proportional dependence between the content of SSB-proteins in chromatin and the intensity of DNA synthesis; 4) the dependence between the content of SSB-proteins in the cells and genomic size in different organisms; 5) the capacity of SSB-proteins to specific stimulation of DNA replicative synthesis. Apparently SSB-proteins in chromatin modulate the replication with the aid of the unwinding of DNA, stabilizing of the single-strand region in this molecule and the activation of the DNA synthesis carried out by DNA polymerase.

Animals↗
PubMed2001

Tuning DNA "strings": modulating the rate of DNA replication with mechanical tension.

Recent experiments have measured the rate of replication of DNA catalyzed by a single enzyme moving along a stretched template strand. The dependence on tension was interpreted as evidence that T7 and related DNA polymerases convert two (n = 2) or more single-stranded template bases to double helix geometry in the polymerization site during each catalytic cycle. However, we find structural data on the T7 enzyme--template complex indicate n = 1. We also present a model for the "tuning" of replication rate by mechanical tension. This model considers only local interactions in the neighborhood of the enzyme, unlike previous models that use stretching curves for the entire polymer chain. Our results, with n = 1, reconcile force-dependent replication rate studies with structural data on DNA polymerase complexes.

Bacteriophage T7↗
PubMed1982

Purification of an adenovirus-coded DNA polymerase that is required for initiation of DNA replication.

Temperature-sensitive mutants in the N complementation group of human adenovirus type 5 are defective at the nonpermissive temperature for replication of virus DNA and for transformation of rat embryo cells. We show that nuclear extracts prepared from Ad5ts 149-infected cells grown at the nonpermissive temperature fail to replicate DNA in vitro. The defect lies in the first step in the initiation of viral DNA synthesis, the formation of a covalent linkage between the terminal protein precursor (pTP) and dCMP. A 140 kilodalton (140 kd) protein which complements these defective extracts and contains DNA polymerase activity has been purified from HeLa cells infected with wild-type Ad2. It is tightly associated with the 80 kd pTP in a replication complex. Both of these proteins are products of the E2B region of the adenovirus genome, and the 140 kd protein coding sequences lie immediately downstream from those encoding the 80 kd protein. These results demonstrate that adenovirus encodes a novel DNA polymerase that is required for priming of DNA synthesis at the origin of replication. This protein may also function in the initiation of transformation of cultured cells.

Adenoviruses, Human↗
PubMed1999

Initiation of DNA replication in eukaryotes: questioning the origin.

Although proteins involved in DNA replication in yeast have counterparts in multicellular organisms, the definition of an origin of DNA replication and its control in higher eukaryotes might obey to different rules. Origins of DNA replication that are site-specific have been found, supporting the notion that specific DNA regions are used to initiate DNA synthesis along metazoan chromosomes. However, the notion that specific sequences will define origins is still being debated. The variety and complexity of transcriptional programs that have to be regulated in multicellular organisms may impose a plasticity that would not be compatible with a fixed origin simply defined at the sequence level. Such a plasticity would be essential to developmental programs where the control of DNA replication could be more integrated to the control of gene expression than in unicellular eukaryotes.

Animals↗
PubMed1995

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication.

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

3T3 Cells↗