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At least 1,243 records · Page 69Linked to original sources
PubMed1999

DNA replication in the 1-cell mouse embryo: stimulatory effect of histone acetylation.

Temporal and spatial distribution of the sites of DNA replication were examined in 1-cell mouse embryos. Embryos were labelled with bromodeoxyuridine (BrdU) at hourly intervals after fertilisation, and the incorporation of BrdU was examined by laser-scanning confocal microscopy following immunostaining with an anti-BrdU antibody. DNA replication first started uniformly in both the male and female pronuclei in the intranuclear region and then was observed in the peripheral regions of nucleus and nucleolus. These changes, however, occurred asynchronously in that the female pronucleus required a longer time to complete replication in the intranuclear region but not in the peripheral regions. Inhibiting transcription with alpha-amanitin had no effect on the temporal and spatial patterns of DNA replication. Treatment of the embryos with trapoxin, a specific inhibitor of histone deacetylase, accelerated the completion of replication in the peripheral regions but not in the intranuclear region. These results suggest that DNA replication is temporally and spatially regulated in the 1-cell embryos and that acetylation of histones, but not transcription, is involved in the regulation of DNA replication.

Acetylation↗
PubMed1990

Exposure to camptothecin breaks leading and lagging strand simian virus 40 DNA replication forks.

To better understand aberrant simian virus 40 DNA replication intermediates produced by exposure of infected cells to the anticancer drug camptothecin, we compared them to forms produced by S1 nuclease digestion of normal viral replication intermediates. All of the major forms were identical in both cases. Thus the aberrant viral replicating forms in camptothecin-treated cells result from DNA strand breaks at replication forks. Linear simian virus 40 forms which are produced by camptothecin exposure during viral replication were identified as detached DNA replication bubbles. This indicates that double strand DNA breaks caused by camptothecin-topoisomerase I complexes occur at both leading and lagging strand replication forks in vivo.

Animals↗
PubMed1978

Simian virus 40 DNA replication in isolated replicating viral chromosomes.

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.

Cell Line↗
PubMed1969

The relationship of DNA replication to the control of protein synthesis in protoplasts of T4-infected Escherichia coli B.

The relationship of DNA replication to the control of early enzyme synthesis and the formation of late proteins was studied in protoplasts of E. coli B cells infected with an amber mutant, T4am130. In this phage-host system the bacterial DNA is not completely degraded, and the onset and the extent of phage DNA synthesis could be regulated by use of 5-fluorodeoxyuridine and thymidine. It was found that DNA replication is necessary for the transcription of "late" regions of the phage genome and, thus, for the synthesis of late proteins. In addition, the sustained synthesis of late RNA and the tail-fiber protein was shown to require the continued replication of DNA. Furthermore, the mechanism regulating the cessation of early enzyme synthesis appeared to become operative shortly after the onset of DNA replication and required a limited amount of DNA synthesis for its full expression.

Carbon Isotopes↗
PubMed1999

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

DNA Repair↗
PubMed1988

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro.

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.

Adenoviridae↗
PubMed1997

RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

DNA Primers↗
PubMed1992

Characterization of linker insertion and point mutations in the NS-1 gene of minute virus of mice: effects on DNA replication and transcriptional activation functions of NS-1.

The NS-1 gene of minute virus of mice encodes a multifunctional protein required for replication of the viral genome and for transcriptional regulation of the two MVM promoters. To study the localization of activities required for DNA replication and transactivation of the capsid gene promoter, insertion and point mutations were introduced into the NS-1 gene. The mutant NS-1 genes were expressed in COS-7 cells by using an SV 40 promoter driven NS-1 expression vector. The ability of the mutant proteins to complement a replication defective NS-1 mutant of the infectious MVM plasmid pMM984 and to activate transcription from the capsid gene promoter in chloramphenicol acetyl transferase expression assays was determined. Two point mutations Ser-249 to Ala and Lys-250 to Gln and a one amino acid insertion between Asp-606 and Leu-607 had no effect on viral DNA replication and transactivation activities. Six independent insertions of between 2 and 12 amino acids inhibited the DNA replication activity of NS-1 between 20- and at least 100-fold. There was no apparent correlation between the extent of inhibition of parvoviral DNA replication and the location of the mutations. The transcriptional activation function of NS-1 was inhibited between 1.5- and at least 20-fold and was therefore overall relatively less sensitive to mutagenesis than was its DNA replication function. An exception to this was a 5 amino acid insertion between Tyr-543 and Gln-544 that abolished transactivation as well as the ability of NS-1 to complement viral DNA replication.

Amino Acid Sequence↗
PubMed1992

The level of supercoiling affects the regulation of DNA replication in Escherichia coli.

The chromosome of Escherichia coli is negatively supercoiled. This favours processes that unwind the two DNA strands, such as DNA replication. In this paper, we have investigated the effect of changed levels of overall chromosomal supercoiling on the initiation of DNA replication. Specifically, we have used flow cytometry to reveal effects on the synchrony of initiations of DNA replication in single cells. An increase in the level of supercoiling moderately reduced initiation synchrony. In contrast, decreased supercoiling led to pronounced asynchrony. We have excluded the possibility that this asynchrony is caused by changes in the level of the Dam methyltransferase or the DnaA protein. We suggest that the global level of supercoiling influences the topology of oriC and thereby the sequence of events leading to initiation of DNA replication in E. coli.

Bacterial Proteins↗
PubMed1978

Strand-specific break near the origin of bacteriophage P2 DNA replication.

Membrane-associated P2 DNA isolated early after infection under conditions that block replication (amB in phage and rep in Escherichia coli C) was analyzed by electron microscopy. Most DNA was in the form of relaxed circles (40%) and circles with short single-stranded tails (60%). When this DNA was hybridized with separate strands of linear P2 Hy dis DNA (which provides suitable reference points along the heteroduplex molecules), an interruption was located near the previously mapped origin of P2 DNA replication in one specific strand. The same strand was sometimes extended in the direction consistent with the unidirectional mode of P2 DNA replication. Similar conclusions were reached when the intracellular DNA was analyzed after partial denaturation. These results are consistent with the rolling circle mode of DNA replication.

Chromosome Mapping↗
PubMed2002

High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

Animals↗
PubMed2002

Control of complex formation of DNA polymerase alpha-primase and cell-free DNA replication by the C-terminal amino acids of the largest subunit p180.

DNA polymerase alpha-primase is a heterotetrameric complex essential for simian vacuolating virus 40 (SV40) DNA replication. We show that the C-terminal 67 amino acid residues of the human p180 subunit are essential for SV40 DNA replication as they are required for binding of the p68 subunit and play a role in the interaction with the primase subunits, p48 and p58. Furthermore, we demonstrate that exchanging these residues to those of mouse origin can only partially rescue the SV40 DNA replication activity of DNA polymerase alpha-primase.

Animals↗
PubMed2002

Proliferating cell nuclear antigen associates with histone deacetylase activity, integrating DNA replication and chromatin modification.

Faithful inheritance of the chromatin structure is essential for maintaining the gene expression integrity of a cell. Histone modification by acetylation and deacetylation is a critical control of chromatin structure. In this study, we test the hypothesis that histone deacetylase 1 (HDAC1) is physically associated with a basic component of the DNA replication machinery as a mechanism of coordinating histone deacetylation and DNA synthesis. Proliferating cell nuclear antigen (PCNA) is a sliding clamp that serves as a loading platform for many proteins involved in DNA replication and DNA repair. We show that PCNA interacts with HDAC1 in human cells and in vitro and that a considerable fraction of PCNA and HDAC1 colocalize in the cell nucleus. PCNA associates with histone deacetylase activity that is completely abolished in the presence of the HDAC inhibitor trichostatin A. Trichostatin A treatment arrests cells at the G(2)-M phase of the cell cycle, which is consistent with the hypothesis that the proper formation of the chromatin after DNA replication may be important in signaling the progression through the cell cycle. Our results strengthen the role of PCNA as a factor coordinating DNA replication and epigenetic inheritance.

Cell Cycle↗
PubMed1982

[DNA replication in adult and aged rat tissues].

The incorporation of single-stranded DNA replication intermediates (Okazaki fragments) into double-strand polymerized DNA and the chain growth of these intermediates in adult and aged rat tissues with low and high mitotic activities were studied. These processes were found to proceed in a similar manner independently of the tissue mitotic activity. However, in tissues with a low mitotic activity of adult and aged rats the incorporation of Okazaki fragments into double-stranded DNA was decelerated as compared to those with a high mitotic activity. Since the mitotic activity and ageing were shown to have similar effects on the rate of stabilization of replicating DNA secondary structure, it may be assumed that the factors responsible for regulation of DNA replication rate are altered with ageing.

Aging↗
PubMed1999

DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase.

CDC7 is an essential gene required for DNA replication in Saccharomyces cerevisiae. Cdc7p homologs have recently been identified in vertebrates, but their role in DNA replication has not yet been addressed. Here we show that antibodies to the Xenopus laevis homolog, xCdc7, interfere with DNA replication in vivo in developing embryos and in vitro in cycling egg extracts. We also demonstrate cell cycle-dependent association of xCdc7 with the Mcm complex, which binds to replication origins and also is required for DNA synthesis. Taken together, these data indicate that the function of xCdc7 is conserved from fungi to vertebrates. xCdc7 protein accumulates after stimulation of resting oocytes with progesterone, suggesting a molecular explanation for previous observations that the development of the capacity for DNA replication requires protein synthesis late in meiosis I.

Amino Acid Sequence↗
PubMed1996

cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.

Cell Cycle↗
PubMed1990

A tumor promoting phorbol ester, TPA, enhances polyomavirus DNA replication by activating the function of the viral enhancer.

The effect of tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) on enhancer dependent polyomavirus (Py) DNA replication was examined in cells expressing Py large T antigen. The results showed that TPA enhanced Py DNA replication by stimulating the activity of the A element, one of the two cores of Py enhancer. Of the three subdomains of the A element, the biding sites of PEBP1 (PEA1), a member of AP1 family, and of PEBP5 were by themselves able to activate Py DNA replication. Furthermore, each binding site of PEBP1 and PEBP5 responded to TPA to enhance Py DNA replication. The results suggest that growth promoting signals could activate DNA replication directly via enhancer binding proteins.

Antigens, Polyomavirus Transforming↗
PubMed1988

Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III.

Octamer transcription factor-1 (OTF-1) and nuclear factor III (NF-III) are sequence-specific DNA binding proteins that activate transcription and DNA replication, respectively. It is shown here that OTF-1 is physically and biologically indistinguishable from NF-III. This conclusion is based on the following observations. First, the two proteins have identical mobilities by SDS-polyacrylamide gel electrophoresis. Second, OTF-1 binds to the adenovirus origin of DNA replication at the same site and with the same affinity as NF-III. Third, OTF-1 can substitute for NF-III in activating the initiation of adenovirus DNA replication in vitro. Fourth, the ability of OTF-1 to stimulate viral DNA replication is dependent on the presence of an intact NF-III binding site within the origin of replication. Fifth, NF-III can substitute for OTF-1 in activating in vitro transcription from the human histone H2b promoter. These data suggest the possibility that NF-III/OTF-1 is a protein that functions in both cellular DNA replication and transcription.

Adenoviridae↗