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PubMed1985

Inhibition of DNA replication and DNA polymerase alpha activity by monoclonal anti-(DNA polymerase alpha) immunoglobulin G and F(ab) fragments.

The effect of monoclonal anti-(DNA polymerase alpha) immunoglobulin G (IgG) and F(ab) fragments on DNA replication in lysolecithin-permeabilized human cells and on DNA polymerase alpha activity was determined. DNA polymerase alpha activity in vitro was inhibited equally by the same concentrations of monoclonal IgGs and F(ab) fragments. However, the IgGs and F(ab) fragments were not equally potent in inhibiting DNA replication in permeable cells. In general, the F(ab) fragments were approximately equal to 10-fold more potent than IgGs in inhibiting DNA replication, suggesting the F(ab) fragments cross the nuclear membrane more readily than IgGs. Immunocytochemical studies demonstrated that at least a fraction of anti-(DNA polymerase alpha) IgGs entered the nucleus of permeable cells. For most antibodies tested, the IgG or F(ab) concentration needed to inhibit replication was several orders of magnitude higher than that needed to neutralize polymerase alpha activity extracted from the same number of cells. Anti-(DNA polymerase alpha) F(ab) fragments were shown to inhibit the discontinuous synthesis of Okazaki DNA, as well as the maturation of Okazaki DNA to larger DNA, thereby implicating DNA polymerase alpha in both of these processes.

Animals↗
PubMed1979

Dependence of mammalian DNA replication on DNA supercoiling. II. Effects of novobiocin on DNA synthesis in Chinese hamster ovary cells.

Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.

Animals↗
PubMed1978

Replication process of the parvovirus H-1. X. Isolation of a mutant defective in replicative-form DNA replication.

A temperature-sensitive mutant of H-1, ts14, that is partially defective in replicative-form (RF) DNA synthesis has been isolated. ts14 H-1 is characterized by a decrease in plaque-forming ability and production of infectious virus at the restrictive temperature of 39.5 degrees C. RF DNA synthesis of ts14 is reduced to 3 to 7% of that of wild-type H-1 at either the restrictive or the permissive temperature. A complementation analysis of RF synthesis of ts14 and a viable defective H-1 virus, DI-1, or wild-type H-3 indicates that the defective RF DNA synthesis of ts14 is cis-acting. ts14, unlike wild-type H-1, causes a multiplicity-dependent inhibition of DI-1 or H-3, but not LuIII, RF DNA synthesis. Mixed infections of cells with two parvoviruses also exhibited a cross-interference for viral protein synthesis that was multiplicity dependent, ts14 inhibited infectious virus production of H-1 or H-3, but not LuIII. LuIII-or H-3-pseudotype particles were produced by coinfection with H-1. H-3 and H-1 showed similar interactions with ts14, and H-3 DNA was more homologous to H-1 than was LuIII by comparative physical mapping studies. The results suggest that ts14 is a mutant with a defect in a regulatory sequence of its DNA that influence RF DNA replication.

DNA Replication↗
PubMed1993

Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli.

Escherichia coli cells induced for the SOS response express inducible stable DNA replication (iSDR) as an SOS function. Initiation of iSDR is independent of transcription, translation and DnaA protein, which are essential for initiation of DNA replication from oriC. We found that a recA mutant that is defective in recombination but proficient in SOS induction could not elicit iSDR. In contrast, iSDR was enhanced by recD and recJ mutations that inactivate the exonuclease V activity of the RecBCD enzyme and the RecJ exonuclease activity, respectively. A mutation in the ruvC gene that blocks the resolution of recombination intermediates (i.e. Holliday structures) also enhanced iSDR. Furthermore, inhibition of branch migration by recG or ruvAB mutations dramatically increased the iSDR activity. recBC mutants are defective in iSDR induction but the defect was suppressed by a mutation in the sbcA gene. The major product of minichromosomes replicated by iSDR was covalently closed circular monomers. We propose that recombination intermediates (i.e. D-loop structures) created by the action of RecA recombinase and RecBC(D) helicase play a central role in initiation of iSDR.

Bacterial Proteins↗
PubMed1999

Nuclear import of p53 during Xenopus laevis early development in relation to DNA replication and DNA repair.

The role of p53 in transcriptional activation of genes involved in cell cycle progression is well established. However, the wide range of functions attributed to this gene suggests that some of them might be unrelated to transcription. Here we investigated p53 localization and recruitment to chromatin during Xenopus early development when 12 rapid cell cycles occur without transcription of the genome. We show that after fertilization, part of the large store of p53 previously stored in the cytoplasm of the oocyte is imported into the nucleus. This import was further analyzed in relation with DNA replication and DNA repair using cell-free systems from Xenopus eggs. Formation of a nuclear lamina envelope is necessary for the import of p53 into the nucleus. p53 associates both with decondensed DNA and the nuclear lamina envelope, but no colocalization with prereplication or replication complexes is observed. We show that UV- or gamma-damaged nuclei recruit p53 as well as replication protein A (RPA) in large common foci. Together, these data suggest that p53 plays a role in the regulation of the accelerated S phases that occur during Xenopus early development, in a manner that does not rely on its transcription-mediated activity.

Animals↗
PubMed1986

Synthesis of bacteriophage phi X174 in vitro: mechanism of switch from DNA replication to DNA packaging.

Replication of a replicative form DNA of bacteriophage phi X174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA. Gene C protein of phi X174, which is involved in DNA packaging, inhibits the rolling-circle DNA synthesis by binding to the initiation complex in vitro. The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with phi X174 gene J protein and the prohead. Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex. These results indicate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage capsid, which occurs in the late stage of infection.

Bacteriophage phi X 174↗
PubMed1997

A role for DNA primase in coupling DNA replication to DNA damage response.

The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.

Blotting, Western↗
PubMed1981

An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles.

Animals↗
PubMed2004

Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage.

Cell cycle arrest in response to environmental effects can lead to DNA breaks. We investigated whether inhibition of DNA replication during the initiation step can lead to DNA damage and characterised a cell-cycle-arrest point at the replication initiation step before the establishment of active replication forks. This arrest can be elicited by the iron chelators mimosine, ciclopirox olamine or 2,2'-bipyridyl, and can be reversed by the removal of the drugs or the addition of excess iron. Iron depletion induces DNA double-strand breaks in treated cells, and activates a DNA damage response that results in focal phosphorylation of histone H2AX, focal accumulation of replication protein A (RPA) and ATR (ATM and Rad3-related kinase), and activation of CHK1 kinase. Abrogation of the checkpoint response does not abolish the cell cycle arrest before the establishment of active DNA replication forks. DNA breaks appear concomitantly with the arrival of cells at the arrest point and persist upon release from the cell cycle block. We conclude that DNA double-strand breaks are the consequence, and not the cause, of cell cycle arrest during the initiation step of DNA replication by iron chelation.

2,2'-Dipyridyl↗
PubMed2001

Regulation of dna replication after heat shock by replication protein a-nucleolin interactions.

Heat shock inhibits replicative DNA synthesis, but the underlying mechanism remains unknown. We investigated mechanistic aspects of this regulation in melanoma cells using a simian virus 40 (SV40)-based in vitro DNA replication assay. Heat shock (44 degrees C) caused a monotonic inhibition of cellular DNA replication following exposures for 5-90 min. SV40 DNA replication activity in extracts of similarly heated cells also decreased after 5-30 min of exposure, but returned to near control levels after 60-90 min of exposure. This transient inhibition of SV40 DNA replication was eliminated by recombinant replication protein A (rRPA), suggesting a regulatory process targeting this key DNA replication factor. SV40 DNA replication inhibition was associated with a transient increase in the interaction between nucleolin and RPA that peaked at 20-30 min. Because binding to nucleolin compromises the ability of RPA to support SV40 DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock. The relevance of this interaction to the regulation of cellular DNA replication is indicated by the transient translocation in heated cells of nucleolin from the nucleolus into the nucleoplasm with kinetics very similar to those of SV40 DNA replication inhibition and of RPA-nucleolin interaction. Because the targeting of RPA by nucleolin in heated cells occurs in an environment that preserves the activity of several essential DNA replication factors, active processes may contribute to DNA replication inhibition to a larger degree than presently thought. RPA-nucleolin interactions may reflect an early step in the regulation of DNA replication, as nucleolin relocalized into the nucleolus 1-2 h after heat exposure but cellular DNA replication remained inhibited for up to 8 h. We propose that the nucleolus functions as a heat sensor that uses nucleolin as a signaling molecule to initiate inhibitory responses equivalent to a checkpoint.

DNA Replication↗
PubMed1998

Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase alpha-primase.

The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). Fluorescence titrations revealed noncompetitive inhibition of TNP-ADP binding by origin DNA, and noncompetitive inhibition of TNP-ADP and TNP-ATP binding by pol alpha, suggesting that T antigen complexed with either origin DNA or pol alpha was not able to bind the TNP nucleotide. From these titrations, we have measured a binding stoichiometry of 11.5 +/- 0.8 T antigen monomers per viral origin DNA, in agreement with the double hexamer assembly of T antigen on the origin as reported earlier. The stoichiometry of pol alpha binding to T antigen was measured to be 5.5 +/- 0.6 mol of T antigen per mole of pol alpha. While monomeric T antigen-nucleotide complex was a preferred ligand over free T antigen in the double hexamer assembly reaction, preformed T antigen hexamers were incapable of forming double hexamers on the DNA. The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.

Antigens, Polyomavirus Transforming↗
PubMed2001

Initiation of DNA replication by DNA polymerases from primers forming a triple helix.

Despite extensive studies on oligonucleotide-forming triple helices, which were discovered in 1957, their possible relevance in the initiation of DNA replication remains unknown. Using sequences forming triple helices, we have developed a DNA polymerisation assay by using hairpin DNA templates with a 3' dideoxynucleotide end and an unpaired 5'-end extension to be replicated. The T7 DNA polymerase successfully elongated nucleotides to the expected size of the template from the primers forming triple helices composed of 9-14 deoxyguanosine-rich residues. The triple helix-forming primer required for this reaction has to be oriented parallel to the homologous sequence of the hairpin DNA template. Substitution of the deoxyguanosine residues by N7 deazadeoxyguanosines in the hairpin of the template prevented primer elongation, suggesting that the formation of a triple helix is a prerequisite for primer elongation. Furthermore, DNA sequencing could be achieved with the hairpin template through partial elongation of the third DNA strand forming primer. The T4 DNA polymerase and the Klenow fragment of DNA polymerase I provided similar DNA elongation to the T7 polymerase-thioredoxin complex. On the basis of published crystallographic data, we show that the third DNA strand primer fits within the catalytic centre of the T7 DNA polymerase, thus underlying this new property of several DNA polymerases which may be relevant to genome rearrangements and to the evolution of the genetic apparatus, namely the DNA structure and replication processes.

Bacteriophage T7↗
PubMed2002

A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication.

DNA polymerases that initiate replication by protein-priming are able to catalyze terminal protein (TP)-primed initiation, the following transition steps and finally DNA-primed elongation. Therefore, their structures must be able to position sequentially both primers, TP and DNA, at a common binding site. For DNA-templated initiation, these DNA polymerases have to bind the origin of replication as template and TP as primer. It is likely that very precise interactions are required to position both TP and templating nucleotide at the polymerization active site. Such a specificity during TP-priming must rely on specific amino acids that must be evolutionarily conserved in this subfamily of DNA polymerases. By site-directed mutagenesis, we have analyzed the functional significance of Lys392 of phi29 DNA polymerase, immediately adjacent to the Kx3NSxYG motif, and specifically conserved among protein-primed DNA polymerases. During TP-primed initiation, mutations in this residue did not affect untemplated TP-dAMP formation, indicating that the interaction with the initiating nucleotide and TP were not affected, whereas the template-directed initiation activity was severely inhibited. Both mutant DNA polymerases had a wild-type-like (overall) DNA binding activity. We thus infer that residue Lys392 of phi29 DNA polymerase is important for the correct positioning of the templating nucleotide at the polymerization active site, a critical requirement during template-directed TP-priming at phi29 DNA origins. Consequently, mutation of this residue compromised the fidelity of the initiation reaction, not controlled by the 3'-5' exonuclease activity. During DNA-primed polymerization, the mutant polymerases showed a defect in translocation of the template strand. This translocation problem could be the consequence of a more general defect in the stabilization and positioning of a next templating nucleotide at the polymerization active site, during DNA-primed DNA synthesis.

Amino Acid Sequence↗
PubMed1993

DNA replication in fertilized eggs of the starfish Asterina pectinifera.

Early starfish embryos were labeled with 5-bromo-2'-deoxyuridine (BrdU; the thymidine analogue) for 3-4 min of pulse, and the BrdU incorporated into newly replicated DNA was immunocytochemically detected with anti-BrdU antibody. Series of optical sections of the stained embryos observed with a confocal laser scanning microscope (CLSM) were collected, and three-dimensional images of the chromatin and the incorporated BrdU were reconstructed on a computer display. The distribution of the DNA replication sites and the chromatin structure were investigated from a three-dimensional point of view. The oocytes fertilized during maturation and the eggs fertilized after maturation were examined for the timing of S phase, DNA replication pattern, and chromatin distribution in the nucleus. Duration of the first S phase between the two types of embryos differed in the first S phase, but was the same in the second and third S phases. We observed a successive change in the distribution of DNA replication sites from a "uniform" to a "condensing" pattern during a single S phase traverse, except in the first S phase of the eggs fertilized after maturation. These two patterns of DNA replication almost coincided with chromatin visualized by anti-single-stranded DNA antiserum. The apparent change in the pattern of DNA replication sites thus simply reflects the change in the spatial distribution of chromatin. The spatial pattern of DNA replication at the first S phase of the eggs fertilized after maturation was unique in that the whole nucleus was a mosaic of two distinct regions, each supposedly derived from the female and male pronuclei. The putative female region had a fibrillar pattern of DNA replication sites confined to the periphery of the nucleus, while the male region showed condensed sites of DNA replication, which persisted longer than the sites in the female pronuclear region. We discuss such a singularity of the first S phase in starfish eggs on the basis of the temporal relation between the progress of maturation and the time of activation.

Animals↗
PubMed2002

Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: translesion DNA synthesis, nucleotide insertion, and extension kinetics.

2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3'-5' exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f(ins) = V(max)/K(m) for insertion) and extension (f(ext) = V(max)/K(m) for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f(ins) = 1) dTMP (2.4 x 10(-4)) approximately dAMP (8.1 x 10(-5)) > dGMP (4.5 x 10(-7))), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f(ext) = 1) > mFapyG:T (4.6 x 10(-3)) mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.

Base Sequence↗
PubMed1990

An abnormal profile of DNA replication intermediates in Bloom's syndrome.

Bloom's syndrome (BS) cells display a characteristic genomic instability, notably an elevated frequency of sister-chromatid exchange. Replicating DNA in cultured BS cells was labeled with [3H]thymidine using several time schedules. Separation of DNA in agarose gels showed high molecular weight DNA and three classes of DNA replication intermediates: 20-kilobase DNA, 10-kilobase DNA, and Okazaki fragments. In contrast newly replicated DNA from normal cells showed no 20-kilobase DNA replication intermediates. Certain BS cells, exceptional in that their characteristic genomic instability has for unknown reasons been corrected, also differed from normal cells in having the 20-kilobase intermediate, but they differed from both normal cells and the other (the uncorrected) BS cells in lacking the 10-kilobase DNA replication intermediates.

Aphidicolin↗
PubMedDate not supplied

[Stimulation of replicative DNA synthesis by eukaryotic proteins bound to single-stranded DNA].

The paper deals with the effect of the single-strand (ss) DNA-binding proteins (SSB-proteins) from the Ehrlich ascites tumor (EAT) cells and from the eggs of silkworm, as well as the mouse serum blood proteins, having preferential affinity to ss DNA, on the DNA replicative synthesis in the EAT cells permeable for the macromolecules, and, for the silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm proteins and on the DNA replicative synthesis in the nuclei from the eggs of silkworm permeable for macromolecules. SSB-proteins of EAT to considerable extent stimulated the DNA synthesis. At the same time, the other proteins (from the silkworm and from the serum) activated the DNA synthesis in the permeable cells to the less extent. It was found that SSB-proteins from the silkworm had a 1.5-13 fold stimulating effect on the DNA replicative synthesis in the homologous system (in the permeable nuclei). If the permeability for the macromolecules of the cells and nuclei treatment with Triton X-100 may be different, it is supposed that the activation of the DNA synthesis by the exogenous proteins depends on the homologous system of the DNA replicative complex. It is possible that the effect of the serum proteins on the DNA synthesis is connected with the masking of the ss regions of DNA which inhibited DNA-polymerase alpha. Perhaps the mechanisms of the activation of the DNA replicative synthesis by the proteins in vitro with the purified DNA polymerase alpha and in vivo are of different nature and are conditioned by homology of the deoxyribonucleoproteins.

Animals↗