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At least 1,279 records · Page 71Linked to original sources
PubMed1985

Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.

Animals↗
PubMed2004

Regulation of eukaryotic DNA replication by proliferation associated protein, PA2G4, in vitro.

To search a novel factor possibly involved in the regulation of DNA replication for eukaryotic cells, we fractionated the Xenopus egg extract frequently used as a cell free DNA replication system, and obtained a single polypeptide of 50 KDa on SDS-polyacrylamide gel electrophoresis. Data of the amino acid sequence reduced from cloned gene of this protein revealed that this might be a Xenopus homolog of human PA2G4 that was known as a proliferation-associated protein or a cell cycle-regulated protein. Recombinant mouse PA2G4 protein also showed the inhibitory effect on the DNA replication reaction in Xenopus egg extract with the single-stranded DNA template. These results strongly suggest that PA2G4 may have an important role on the regulation of DNA replication in eukaryotic cells.

Animals↗
PubMedDate not supplied

Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae.

A detailed characterization of the mak1-3 mutation of Saccharomyces cerevisiae has been made possible by modifying its genetic background. The mak1-3 mutation, which confers temperature sensitivity for growth, was originally identified as one of four mak1 mutations (Wickner and Leibowitz, 1976). Mak1-1, 1-2 and 1-4 mutants are deficient in DNA topoisomerase I activity and thus have been renamed 'top1' (Thrash et al., 1984). Studies presented here show that the map position of MAK1-3 on chromosome XVI distinguishes it from TOP1 which maps on chromosome XV (Wickner and Leibowitz, 1976). An investigation of in vivo macromolecular synthesis in the mak1-3 mutant shows that it is deficient in DNA replication at the restrictive temperature. Experiments in which DNA synthesis was measured in synchronized cell populations indicate that the mak1-3 mutant is deficient in the initiation step of DNA synthesis. Furthermore, crude extracts from the mak1-3 mutant cells support temperature-sensitive in vitro DNA synthesis on yeast chromosomal DNA replication origin containing plasmid pARS1, suggesting that the MAK1 gene product is directly required for in vitro DNA replication. The conclusion that mak1-3 is a newly identified DNA replication mutation is based on the observations that it (1) complements all DNA synthesis mutants examined, (2) maps to a previously undetected chromosomal location and (3) has a distinct terminal morphology. In light of these distinctions and of the role mak1-3 plays in DNA replication, it has been renamed 'dna1'.

Chromosome Mapping↗
PubMed2004

Plasticity of DNA replication initiation in Epstein-Barr virus episomes.

In mammalian cells, the activity of the sites of initiation of DNA replication appears to be influenced epigenetically, but this regulation is not fully understood. Most studies of DNA replication have focused on the activity of individual initiation sites, making it difficult to evaluate the impact of changes in initiation activity on the replication of entire genomic loci. Here, we used single molecule analysis of replicated DNA (SMARD) to study the latent duplication of Epstein-Barr virus (EBV) episomes in human cell lines. We found that initiation sites are present throughout the EBV genome and that their utilization is not conserved in different EBV strains. In addition, SMARD shows that modifications in the utilization of multiple initiation sites occur across large genomic regions (tens of kilobases in size). These observations indicate that individual initiation sites play a limited role in determining the replication dynamics of the EBV genome. Long-range mechanisms and the genomic context appear to play much more important roles, affecting the frequency of utilization and the order of activation of multiple initiation sites. Finally, these results confirm that initiation sites are extremely redundant elements of the EBV genome. We propose that these conclusions also apply to mammalian chromosomes.

DNA Replication↗
PubMed1993

Fidelity of DNA replication by extracts of normal and malignantly transformed human cells.

To test the hypothesis that a mutator phenotype may be associated with carcinogenesis (L. A. Loeb, Cancer Res., 51: 3074-3079, 1991), we have compared the fidelity of double-stranded DNA replication and the efficiency of mismatch repair in extracts from normal diploid and malignantly transformed human cells. Included was a diploid fibroblast strain and its transformed derivative, as well as a second diploid fibroblast strain and HeLa cells. The fidelity of DNA replication by cytoplasmic extracts in the presence of simian virus 40 large tumor antigen (SV40 T-antigen) was measured using a forward mutagenesis assay. The replicated DNA consisted of double-stranded M13 mp2 DNA containing the SV40 origin of replication and the lacZ alpha complementation gene as a target sequence for scoring mutations. T-antigen-dependent replication was detected in all cell extracts, with those from transformed cells having the greatest activity. No differences in replication fidelity were detected between normal and transformed cell extracts. Using a heteroduplex containing a G.G mispair, we also detected mismatch repair activity in the cell extracts, including efficient repair in extracts from malignantly transformed cells. While these data do not eliminate the possibility that a mutator phenotype may be associated with carcinogenesis, they do suggest that genetic instability associated with transformation does not involve reduced fidelity of replication of undamaged DNA or reduced mismatch repair efficiency.

Cell Line, Transformed↗
PubMed1976

[Characteristics of DNA replication of the chromosomes of the bone marrow cells in patients with acute leukemia].

A study of the late DNA replication pattern in chromosomes of human acute leucaemia cells revealed a significant diffrence from control. Chromosomes, 2,3 and 4-5 of the acute leucaemia cells finish their DNA replication earlier, and chromosomes 1, 13-15 and 16 later, compared to the control chromosomes. The difference in the pattern of DNA replication between analogous chromosomes of acute leucaemia and donor cells was associated with the discovery of large late-replicating chromatin blocks in the pericentromeric regions of leucaemia cell chromosomes. Some relationship is suggested between the pattern of pericentromeric heterochromatin DNA replication and cell differentiation.

Acute Disease↗
PubMed1975

Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.

The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells in fected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single and double-stranded DNA templates. Analysis of this in vitro system reveals most of the features attributed to in vivo DNA replication: (1) De novo DNA chain initiation is found on a single-stranded DNA template only if ribonucleoside triphosphates are present (as expected for RNA priming of Okazaki pieces on the "lagging" strand of a replication fork). (2) With single-stranded circular DNA as template, synthesis continues for many doublings. The products after extensive synthesis resemble a rolling circle as visualized in the electron microscope, with discontinuous "lagging" strand synthesis generating a long, unbranched double-stranded tail. The fact that all six mutationally identified T4 replication gene products are required for these syntheses suggests the existence of a large multienzyme complex, constituting the T4 replication apparatus.

Chromosome Mapping↗
PubMed1994

Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.

Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.

Adenoviruses, Human↗
PubMed2001

An origin of DNA replication from streptomycete phage phi U1.

A DNA fragment from phage phi U1 containing an origin of DNA replication was identified. This fragment, designated ori, was able to support the maintenance in Streptomyces lividans of a plasmid lacking a functional Gram-positive ori. The sequence of the minimal ori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in this ori fragment indicates that host factors alone were sufficient to initiate replication at ori.

Bacteriophages↗
PubMed1970

6-(p-hydroxyphenylazo)-uracil: a selective inhibitor of host DNA replication in phage-infected Bacillus subtilis.

The azopyrimidine, 6-(p-hydroxyphenylazo)-uracil, inhibits the replication of bacterial DNA selectively, completely, and reversibly, and has no apparent effects on the metabolism of other cellular macromolecules thus far examined. The mechanism of its action has been investigated in uninfected and phage-infected Bacillus subtilis, and the compound appears to be specific for a host function. In cells infected with virulent phage the synthesis of phage DNA proceeds normally, while residual host DNA synthesis is completely blocked. The drug-sensitive host site retains its sensitivity even after partial disruption of the cell by lysozyme treatment.

Azo Compounds↗
PubMed1984

Isolation of human DNA sequences that bind to nuclear factor I, a host protein involved in adenovirus DNA replication.

Nuclear factor I is a 47,000-dalton protein isolated from human HeLa cells that is required for the in vitro replication of adenovirus DNA. This protein was previously shown to bind specifically to nucleotides 17-48 of the left-hand terminus of cloned adenovirus serotype 5 DNA. An in vitro assay for DNA sequences that compete with adenovirus DNA for the binding of nuclear factor I has been developed. With this assay, we have shown specific binding of human DNA sequences to nuclear factor I. Using the DNA binding activity of nuclear factor I, we have isolated and cloned segments of human DNA that bind tightly to this protein. One nuclear factor I binding site is present about every 100,000 base pairs in the HeLa cell genome. The binding of these DNA molecules to nuclear factor I resembles the binding of cloned adenovirus DNA to the protein and is resistant to high ionic strength. The isolation of DNA sequences from HeLa cells that bind specifically to nuclear factor I suggests that this protein interacts with host DNA in vivo.

Adenoviruses, Human↗
PubMed1980

Evidence for direct involvement of T7 RNA polymerase bacteriophage DNA replication.

Experiments in a number of different systems have suggested that the initiation of DNA replication is often dependent upon transcription at the origin of replication. During infection with bacteriophage T7, the T7 genome is transcribed first by the bacterial RNA polymerase and then by a phage-coded enzyme, the product of gene 1. The bacterial enzyme does not appear to be directly involved in the initiation of replication as phage DNA synthesis is not inhibited by rifampin. For testing whether the T7 RNA polymerase plays a role in replication, a T7 gene 1 temperature-sensitive mutant was used, and the RNA polymerase was inactivated at intervals after infection by rapidly raising the temperature of the culture. The experiments indicated that the inactivation of the T7 RNA polymerase caused the cessation of phage DNA synthesis, even at later times during infection when the inhibition of protein synthesis with chloramphenicol had no effect on DNA replication. This suggests that in addition to its role in gene expression, the T7 RNA polymerase plays a direct role in T7 DNA replication.

Chloramphenicol↗
PubMed1993

cis-acting elements in the lytic origin of DNA replication of Epstein-Barr virus.

oriLyt, the cis-acting element of Epstein-Barr virus, mediates viral DNA replication in the lytic phase of the virus's life cycle. Oligonucleotide-directed in vitro mutagenesis of oriLyt plasmids allowed the identification of two noncontiguous components within the complex structure of oriLyt. Both components were indispensable for DNA replication of this origin. The upstream component colocalized with the promoter of the viral BHLF1-encoding gene, and mutants affecting DNA replication affected RNA transcription, too. The second component crucial for oriLyt function was determined to be 40 bp long and positioned approximately 530 bp downstream. It was dispensable for transcriptional transactivation but it was absolutely required for replication. Thus, the overall design of oriLyt has striking similarity to multipartite regulatory elements of transcription, consisting of proximal promoters and distal enhancers, but special elements are exclusively dedicated to DNA replication.

Base Sequence↗
PubMed1993

Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29.

DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal region of approximately 400 bp which lies upstream of the gene that is homologous to herpes simplex virus UL29, a region that carries an origin in other betaherpesviruses and in some alphaherpesviruses.

DNA Mutational Analysis↗
PubMed2003

Correlation between premeiotic DNA replication and chromatin transition at yeast recombination initiation sites.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae are preceded first by DNA replication and then by a chromatin transition at DSB sites. This chromatin transition, detected as a quantitative increase in micrococcal nuclease (MNase) sensitivity, occurs specifically at DSB sites and not at other MNase-sensitive sites. Replication and DSB formation are directly linked: breaks do not form if replication is blocked, and delaying replication of a region also delays DSB formation in that region. We report here experiments that examine the relationship between replication, the DSB-specific chromatin transition and DSB formation. Deleting replication origins (and thus delaying replication) on the left arm of one of the two parental chromosomes III affects DSBs specifically on that replication-delayed arm and not those on the normally replicating arm. Thus, replication timing determines DSB timing in cis. Delaying replication on the left arm of chromosome III also delays the chromatin transition at DSB sites on that arm but not on the normally replicating right arm. Since the chromatin transition precedes DSB formation and requires the function of many genes necessary for DSB formation, these results suggest that initial events for DSB formation in chromatin are coupled with premeiotic DNA replication.

Chromatin↗
PubMed1996

DNA replication of IncQ broad-host-range plasmids in gram-negative bacteria.

Bacterial plasmids of Escherichia coli incompatibility group Q (IncQ) are broad-host-range plasmids that are able to proliferate in almost all Gram-negative bacteria. They are small, nonconjugative, and multicopy plasmids. They can be mobilized into many species of Gram-negative bacteria by coresident conjugative plasmids. Plasmids RSF1010, R1162, and R300B have DNAs of a size of 8.7 kb, and are best studied among IncQ plasmids. These plasmids encode by themselves three major proteins essential for the initiation of DNA replication. This makes the plasmid DNA replication less dependent on the DNA replication apparatus of host cells, and leads to promiscuity or a broad host range. Considering the biological features of these plasmids, they are potent DNA cloning vehicles. Moreover, their characteristic DNA replication mechanism that makes IncQ plasmids promiscuous is elaborate, and is an interesting object of scientific studies.

DNA Replication↗
PubMed2005

Roles of replication fork-interacting and Chk1-activating domains from Claspin in a DNA replication checkpoint response.

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265-605) that associates with Cdc45, DNA polymerase epsilon, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265-331 and 470-600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776-905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1.

Adaptor Proteins, Signal Transducing↗
PubMed1994

The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA.

The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Here we use simian virus 40 DNA replication in vitro to show than p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. Furthermore, p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA.

Cell Division↗