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At least 721 records · Page 40Linked to original sources
PubMed1999

Formation of knots in partially replicated DNA molecules.

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.

DNA Replication↗
PubMed1995

Consequences of 2',2'-difluorodeoxycytidine (gemcitabine) on replicative DNA synthesis in intact HL-60 cells.

The technique of pH-step alkaline elution was used to assess the effects of gemcitabine (dFdC) on replicative DNA synthesis in intact HL-60 human myeloid leukemia cells. Although gemcitabine did cause profound inhibition of DNA chain elongation, it was progressively incorporated through nascent DNA replication intermediates of increasing size into genomic DNA. Hence, in the intact cell, it is not a chain terminator, at least not in the absolute sense of the term. In comparison to cytosine arabinoside (ara-C), the progression of incorporated gemcitabine from small nascent DNA fragments to genomic-length DNA was less complete. Furthermore, at equitoxic exposures, less gemcitabine was incorporated into DNA than ara-C. Studies of the effects of gemcitabine on ribonucleotide reduction in HL-60 cells revealed that dGTP pools, but not dCTP pools, were reduced by a 3-hour exposure to 40 nmol/L gemcitabine (the concentration that causes 50% lethality). This reduction was transient, and recovery of dGTP pool size was accomplished within 16 hours. These studies indicate that the effects of gemcitabine on inhibiting replicative DNA chain elongation comprise an important component of the cytotoxicity of the drug.

Antimetabolites, Antineoplastic↗
PubMed2005

Cell cycle regulation of chromatin at an origin of DNA replication.

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Adenosine Triphosphatases↗
PubMed2004

Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication.

The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites.

Caulobacter crescentus↗
PubMed2004

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication.

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1996

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells.

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.

Adenosine Triphosphate↗
PubMed1978

dnaT, dominant conditional-lethal mutation affecting DNA replication in Escherichia coli.

Normally, bacteria cease DNA replication in the absence of protein synthesis. A variety of treatments, such as thymine starvation or a shift-up to rich medium, lead to continued DNA replication in the absence of protein synthesis. Mutants are described which always terminate replication under these conditions. These conditional lethal mutants, dnaT1 and dnaT2, contransduce with serB and dnaC. The mutation also affects cell division. All aspects of the mutant phenotype (obligatory termination of replication, temperature sensitivity of DNA replication and growth, and aberrant cell division at permissive growth temperatures) were transdominant to the wild-type phenotype. Episomes carrying the dnaT mutation appeared to be unstable. The existence of such a dominant mutation was predicted by a model of chromosome termination proposed by Kogoma and Lark (J. Mol. Biol. 94:243-256, 1975).

Bacterial Proteins↗
PubMed2002

Archaeal DNA replication: spotlight on a rapidly moving field.

The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell. The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like. This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea. Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.

Archaea↗
PubMed2006

Initiation of chromosomal DNA replication in mammalian cell-free systems.

Chromosomal DNA replication is a fundamental part of the cell division cycle of eukaryotes, and its disruption often leads to genome instability and cancer. A focus for regulation is the initiation of the first replication forks, marking the transition from G1 to S phase. Direct biochemical investigation of the establishment and further progression of chromosomal DNA replication in human somatic cell nuclei has become possible through a cell-free system that obeys cell cycle control. Since its development less than a decade ago, several modifications and adaptations of the original system have been reported, which have led to temporal resolution of replication complex assembly and to the identification of novel DNA replication factors. Here, I will review the different systems, highlight fundamental differences and unifying concepts, and discuss their potential for understanding chromosomal DNA replication in somatic mammalian cells.

Animals↗
PubMed1986

Inhibition of adenovirus DNA replication in vitro by autoimmune sera.

Sera from patients suffering from autoimmune diseases were analyzed for the presence of antibodies that inhibit adenovirus DNA replication in vitro. DNA replication was studied in a reconstituted system containing purified viral proteins (DNA binding protein, DNA polymerase and the precursor to the terminal protein) and a crude nuclear extract from HeLa cells. About half the autoimmune sera analyzed inhibited DNA replication by more than 50% while only 2 out of 31 control sera showed strong inhibition. The inhibition was caused by the IgG fractions of the sera and was most frequently observed with sera from scleroderma patients. Several lines of evidence indicate that the inhibition is not due to anti-DNA antibodies. The mechanism of inhibition of two strongly inhibitory sera was further investigated. The IgG fractions from these sera blocked DNA chain elongation more than 80% but had no effect on the initiation step or the synthesis of the first 26 nucleotides. Using a dot blot assay and different incubation conditions, evidence was obtained that the inhibition is due to immunorecognition of a nuclear factor from HeLa cells. Two nuclear proteins are known to be required for adenovirus DNA replication, nuclear factors I and II. DNA replication in the presence of purified nuclear factor I instead of a crude nuclear extract was only slightly inhibited by the antisera. In agreement with this, immunorecognition of nuclear factor I could not be detected using a dot blot assay. Since nuclear factor II is not required in our assay system, these results suggest the existence of another nuclear component involved in adenovirus DNA replication which is neutralized by these antibodies.

Adenoviridae↗
PubMed1994

Topoisomerase IV can support oriC DNA replication in vitro.

Escherichia coli has two type II topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Topo IV is required for the decatenation of the linked daughter chromosomes at the terminal stages of DNA replication, whereas gyrase, because of its ability to convert to negative supercoils the positive supercoils generated by replication fork progression in a circular chromosome, is required to support nascent chain elongation. Using an oriC DNA replication system in vitro, we show that Topo IV, which can relax positive supercoils, can also support replication fork progression. This activity is only observed at substoichiometric ratios of Topo IV to template, at higher ratios, the template becomes relaxed and initiation of DNA replication cannot occur. Topo IV was capable of supporting bidirectional DNA replication from oriC, although, unlike the case with gyrase, some templates apparently replicated unidirectionally. This suggests that either gyrase itself or a certain minimum superhelical density is required for proper initiation of DNA replication from oriC.

Cell-Free System↗
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↗
PubMed2004

Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase.

The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. We show that, during this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Dissociation of the protein primer and DNA polymerase after initiation of adenovirus DNA replication.

Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP.trinucleotide complex (pTP.CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP.pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP.C and pTP. CA were still bound to the polymerase, after the formation of pTP. CAT 60% of the pTP.pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and in Km for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA ... instead of the natural GTAGTA ... ) we show that G5 rather than G3 is used to start, leading to a pTP. tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two.

Adenoviridae↗
PubMed1995

Analysis by pulsed-field gel electrophoresis of DNA double-strand breaks induced by heat and/or X-irradiation in bulk and replicating DNA of CHO cells.

For a given amount of cell killing, heat alone (10-80 min, 45.5 degrees C) induced very few double-strand breaks (dsbs) compared with X-rays. Furthermore, 10 min at 45.5 degrees C immediately prior to X-rays caused only a 1.3-fold increase in the slope of the X-ray-induced dsb dose-response curve, i.e. 0.67 +/- 0.006 (95% confidence) dsbs/100Mbp/Gy for heated cells compared with 0.53 +/- 0.005 for unheated control cells. However, this same heat treatment caused > 5-fold inhibition in the rate of repair of dsbs induced by 60-Gy X-rays, with the degree of inhibition being much less in thermotolerant (TT) cells than in non-tolerant (NT) cells. This reduced inhibition of repair in TT cells correlated with the more rapid removal of excess nuclear protein from nuclei isolated from TT cells than from NT cells. These results plus a TT ratio of 2-3 for both heat-induced radiosensitization and heat-inhibition of repairing dsbs are consistent with the hypothesis that heat radiosensitization results primarily from heat aggregation of nuclear protein interfering with access of repair enzymes to DNA dsbs. The selective heat-radiosensitization of S-phase cells, however, may result from an increase in radiation-induced dsbs in or near replicating regions. For example, a preferential increase in dsbs in replicating DNA compared with bulk DNA was found following either hyperthermia alone (10-30 min, 45.5 degrees C) or a combined treatment (10 min, 45.5 degrees C before 60 Gy). A 30-min treatment at 45.5 degrees C induced dsbs equivalent to approximately 10 Gy in replicating DNA compared with 3-5 Gy in bulk DNA. When cells were heated immediately before irradiation, the increase in dsbs induced in the replicating DNA by 60 Gy was equivalent to 200 Gy. We hypothesize that the observed 2-fold increase in single-stranded regions in replicating DNA after heat resulted in radiation selectively inducing dsbs at or near the replication fork where the heat-induced increase in single-stranded DNA should occur. Thus, this preferential increase in dsbs in the replicating DNA by heat alone and especially when heat was combined with radiation may explain at least in part, the high sensitivity of S-phase cells to heat killing and heat radiosensitization.

Animals↗
PubMed1981

Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

Adenoviruses, Human↗
PubMed1990

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.

Animals↗