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At least 307 records · Page 17Linked to original sources
PubMed1994

Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases.

Polymerases in general share only a few regions of amino acid similarity. One of the most conserved regions, called motif A, has the sequence DXXSLYPSII or a similar sequence in many eukaryotic and viral DNA polymerases and in bacteriophage T4 DNA polymerase. We designed genetic techniques to isolate mutant T4 DNA polymerases with amino acid substitutions in this highly conserved motif. The mutant DNA polymerases differed from wild type T4 DNA polymerase in several ways. For one mutant DNA polymerase, the pyrophosphate analog, phosphonoacetic acid, was a potent inhibitor of DNA replication, and this mutant DNA polymerase replicated DNA with reduced fidelity. Another mutant DNA polymerase replicated DNA with increased accuracy, but this mutant DNA polymerase was less processive in primer extension reactions, and DNA replication required high concentrations of deoxynucleoside triphosphates. We provide evidence that indicates that all of these changes to DNA polymerase function are due to differences in how the mutant DNA polymerases partition between states active for DNA replication or exonucleolytic proofreading. These studies also provide further support for the hypothesis that the accuracy of DNA replication observed for DNA polymerases and 3'-->5' exonuclease activities (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol. Chem. 247, 7116-7122).

Amino Acid Sequence↗
PubMed1993

A new and rapid method for visualising DNA replication in spread DNA by immunofluorescence detection of incorporated 5-iododeoxyuridine.

We have developed a new and rapid immunofluorescent method for visualisation of replicated regions on fixed DNA fibers. Using this method we have found in 5-fluorodeoxyuridine (FrdU)-blocked human cells distinct replication units covering about 60 kb of DNA and corresponding in size to single replicons or chromatin loops. Our results also suggest that nonadjacent replicons within a replicon cluster may be activated after FrdU arrest and that the method may be adapted for localization on fibers of specific DNA sequences.

Chromosomes, Human↗
PubMed1988

Analysis of butylphenyl-guanine, butylphenyl-deoxyguanosine, and butylphenyl-deoxyguanosine triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis using permeable human fibroblasts.

The purine base and nucleoside analogues N2-(p-n-butylphenyl)-guanine (BuPh-Gua) and N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPh-dGuo) are strong inhibitors of isolated mammalian DNA polymerase alpha, but are less potent that expected as inhibitors of DNA replication in intact cultured cells [G. E. Wright, L. W. Dudycz, Z. Kazimierczuk, N. C. Brown and N. N. Khan, J. med. Chem. 30, 109 (1987)]. The mechanistic basis for these observations was explored using permeable human fibroblasts. DNA replication in the permeable cells was inhibited only slightly by BuPh-Gua and BuPh-dGuo at 100 microM, the highest concentration which could be attained. Similar results were obtained for ultraviolet-induced DNA repair synthesis, a process which is though to involve the same DNA polymerase as replication. More detailed studies were performed using the corresponding nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh-dGTP), which is much more water-soluble than the base and nucleoside. The apparent Ki values for BuPh-dGTP inhibition of both replication and ultraviolet-induced repair synthesis in permeable cells were approximately 3 microM. These values are several hundred-fold greater than the apparent Ki for BuPh-dGTP inhibition of isolated human DNA polymerase alpha, which is approximately 10 nM. We conclude that BuPh-Gua and BuPh-dGuo are poor inhibitors of DNA replication in intact cells not because of permeability barriers, but because, unlike polymerase alpha, cellular DNA synthesis is relatively insensitive to this group of inhibitors. These results suggest that polymerase alpha may not be a good general model for predicting the potency of base, deoxyribonucleoside and deoxyribonucleotide analogues as inhibitors of mammalian cellular DNA replication. The fact that the permeable cell systems accurately reflect the relative insensitivity to butylphenyl-guanine derivatives of mammalian DNA replication suggests that permeable cells may be useful tools in future studies of base and nucleoside analogues.

Cells, Cultured↗
PubMed1986

DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate.

In mammalian cells, both semiconservative DNA replication and the DNA repair patch synthesis induced by high doses of ultraviolet radiation are known to be inhibited by aphidicolin, indicating the involvement in these processes of one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. In this paper, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate, a strong inhibitor of polymerase alpha and a weak inhibitor of polymerase delta, is used to further characterize the DNA polymerase(s) involved in these two forms of nuclear DNA synthesis. In permeable human fibroblasts, DNA replication and ultraviolet-induced DNA repair synthesis are more resistant to the inhibitor than DNA polymerase alpha by factors of approximately 500 and 3000, respectively. These findings are most consistent with the involvement of DNA polymerase delta in these processes.

Cells, Cultured↗
PubMed1993

Identification of the initiation region of DNA replication in the murine immunoglobulin heavy chain gene and possible function of the octamer motif as a putative DNA replication origin in mammalian cells.

An origin region of DNA replication in the murine immunoglobulin heavy chain (IgH) gene was identified by BrdU pulse labeling and PCR amplification methods. The origin region spans about 1000 base pairs and contains the region of transcriptional enhancer in which the octamer sequence is present. The octamer sequence, TNATTTGCAT, is a well-conserved promoter/enhancer element responsible for B cell-specific transcription and is also found in the regulatory sequences for histone genes and others. Its activity as an autonomously replicating sequence was further examined. The murine IgH enhancer region containing the octamer motif was cloned in pUC18 and transfected to HeLa cells. After 60-65 h, the low molecular weight DNA was extracted and the degree to which the plasmid DNA had been replicated in the cells was measured by back-transformation of competent bacteria. Five to ten copies of the plasmid were detected per cell. The replicated plasmid-form DNA could be detected by this assay for at least 7 days after transfection. Synthetic oligonucleotides corresponding to the octamer and the Ephrussi box in the IgH enhancer were also cloned into pUC18 and examined for replicating activity. These plasmids replicated provided that the octamer sequence remained intact, irrespective of the Ephrussi box sequence and of the sites of insertion. These results suggest that the octamer transcriptional element may also serve as a putative origin for cellular DNA replication.

Animals↗
PubMed1993

Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system.

The extreme S-phase-specific cytotoxicity of camptothecin has been shown to involve active DNA replication. To investigate the role of DNA replication in camptothecin cytotoxicity, we have studied the interaction between the DNA replication machinery and the topoisomerase I-camptothecin-DNA ternary cleavable complex in a cell-free SV40 DNA replication system. The formation of topoisomerase I-camptothecin-DNA-cleavable complexes on the replication template efficiently and irreversibly inhibited DNA replication. Two aberrant forms of replication products were produced whose abundance varied with the concentrations of exogenously added topoisomerase I and camptothecin. At low concentrations of topoisomerase I and camptothecin, the major aberrant DNA replication product was close-to-unit-length-linear DNA, while at higher concentrations the predominant product was close-to-dimer-size-linear DNA. Analysis of these aberrant replication products has suggested a "collision" model in which the interaction between an advancing replication fork and a topoisomerase I-camptothecin-DNA-cleavable complex results in irreversible arrest of the replication fork and the formation of a double-strand DNA break at the fork. Concomitant with fork arrest and fork breakage, the reversible cleavable complex was converted into a topoisomerase I-linked DNA break. We propose that one or several of these events triggers S-phase-specific cell killing and G2-phase cell cycle arrest.

Camptothecin↗
PubMed1994

Effects of dietary restriction on induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) in rat liver.

The effects of dietary restriction on the induction of unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) were studied in the hepatocytes of F344 rats exposed in vivo to dimethylnitrosamine (DMN) or CCl4. The animals were given food ad libitum, a restricted amount of food (4 g/rat/overnight) or no food. Hepatocytes were isolated 2 h after oral administration of DMN at a dose of 5 mg/kg body weight and 48 h after oral administration of CCl4 at a dose of 400 mg/kg body weight, and incubated for 4 h in Williams' medium E supplemented with either [3H]thymidine for UDS or 5-bromodeoxyuridine for RDS. UDS was determined by autoradiography and RDS was determined by the immunoenzymatic staining method. The background levels of UDS (net grains/nucleus) and RDS (cells in S phase) in control were -12.4 and 0.64% for ad libitum feeding, -6.8 and 0.04% for restricted feeding, and -8.1 and 0% for fasting. UDS induced by DMN and RDS induced by CCl4 were 19.4 and 3.3% for ad libitum feeding, 34.5 and 10.4% for restricted feeding, and 47.8 and 15.1% for fasting. DMN demethylase activity in rat liver was also found to increase with dietary restriction. These results indicate that dietary restriction modulates the responses of UDS and RDS in the liver of rats.

Animals↗
PubMed1984

[DNA replication in mammalian cells exposed to physical, chemical or biological factors. II. DNA-complex and nucleoid recovery and DNA replication after gamma irradiation].

In the irradiated mammalian cells the repair of DNA-membrane complexes (DMC) and nucleoid structure was shown to proceed more quickly than the restoration of DNA synthesis. It is suggested that the repair of DNA-membrane complexes and nucleoid structure is necessary but not sufficient for normalization of DNA replication.

Animals↗
PubMed1987

DNA replication by a DNA-membrane complex extracted from Bacillus subtilis: site of initiation in vitro and initiation potential of subcomplexes.

A DNA-membrane complex extracted from Bacillus subtilis was studied further as a model system for initiation of bacterial DNA replication in vitro. Of three subcomplexes purified from the crude complex by a combination of CsCl and sucrose gradient centrifugation, the synthetic capability of only one was inhibited significantly by streptovaricin, a known inhibitor of RNA primer formation. A selective enrichment in the level of this subcomplex was obtained by manipulating a thymine-requiring mutant. The synthetic capabilities of an enriched and nonenriched DNA-membrane complex were compared in the presence and absence of streptovaricin. Although the rate and extent of DNA synthesis per unit of protein were approximately the same in the absence of the antibiotic, there was a much greater inhibition of synthesis shown by the enriched complex in the presence of streptovaricin. Although the amount of DNA present in the putative initiation subcomplex was less than 0.3 to 0.4% of the total DNA present in the crude complex, such DNA, except for a few quantitative differences, was still representative of genomic DNA. Newly synthesized DNA hybridized to specific origin- and non-origin-derived restriction fragments of the B. subtilis genome. However, when an elongation inhibitor (ddCTP) was added, hybridization of such DNA to almost all of the nonorigin fragments disappeared or was reduced drastically, whereas origin region hybridization patterns remained strong. The highest level of hybridization in the origin region occurred with a BamHI (B7) restriction fragment of 5.6 kilobases that has been implicated by others as one site initiation in vivo (N. Ogasawara, M. Seiki, and H. Yoshikawa, Nature (London) 281:702-704, 1979; S. J. Seror-Laurent and G. Henckes, Proc. Natl. Acad. Sci. USA 82:3586-3590, 1985).

Bacillus subtilis↗
PubMed1992

Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences.

Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.

Animals↗
PubMed1989

Formation and repair of DNA-protein crosslinks in newly replicated DNA.

The production and removal of gamma-radiation-induced DNA-protein crosslinks (DPC) in nuclear matrix-associated newly replicated DNA were examined, as well as the relationship of DPC to DNA replication. In unirradiated, exponentially growing Chinese hamster V79 cells, DNA pulse labeled with [3H]thymidine was observed to be bound preferentially to protein. The pulse-labeled DNA subsequently became dissociated from protein. After a 30- to 60-min chase period, the level of labeled DNA in DPC was reduced to the same level as for bulk DNA. The radiation dose response for the formation of DPC was similar in newly replicated DNA that had been chased for various times and in mature chromatin DNA. Labeled DNA, in the DPC formed after 60 Gy, was rapidly removed from protein during the postirradiation incubation period. However, no recovery of DNA synthesis was observed, even after the majority of DPC were released. Thus either DPC are not the sole cause of the inhibition of DNA synthesis or their removal is not sufficient for DNA synthesis to resume.

Animals↗
PubMed1991

Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.

Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).

Adenosine Triphosphate↗
PubMed2006

Transcription regulatory elements are punctuation marks for DNA replication.

Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as "punctuation marks" for DNA replication in vivo.

AT Rich Sequence↗
PubMed1982

Late events in T4 bacteriophage DNA replication. III. Specificity of DNA reinitiation as revealed by hybridization to cloned genetic fragments.

Through the use of the technique of hybridization to cloned genes, the site specificity of the reinitiation of T4 DNA replication was examined at late times after infection, when a large amount of DNA had accumulated in the infected cell. Replication was examined under two conditions; (i) when there was recombination but the repair of the recombinants was inhibited, and (ii) when recombination was followed by covalent joining. When no covalent repair of recombinant was allowed, reinitiation occurred in the areas known to be also involved in the initiation of replication of the parental molecule: thus late reinitiation, if covalent joining is prevented, is site specific. When there was covalent joining, reinitiation displayed no apparent site specificity. The results are discussed in light of the possibility that at late times after infection recombinant intersections act as primers. The similarity of the model proposed to the "break-and-copy" model for lambda phage and the fitness of the proposed model to the genetic phenomena described by others are emphasized.

Base Sequence↗
PubMed1996

Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.

Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence of numerous in situ DNA strand breaks is also an inherent feature of apoptotic cells. The aim of the present study was to differentially label DNA strand breaks in apoptotic cells vs photolysis-induced breaks in BrdUrd incorporating cells. This would allow one, by multicolor staining, to identify these respective cells in the same sample preparation. Toward this end, exponentially growing HL-60 cells were pulse labeled with BrdUrd and then were subjected to hyperthermia or treated with DNA topoisomerase I inhibitor camptothecin to induce apoptosis. DNA strand breaks in apoptotic cells were first labeled directly with fluorochrome-conjugated dUTP or dCTP, followed by dideoxynucleotide (to terminate chain elongation), in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The cells were subsequently exposed to UV light to photolyze DNA containing the incorporated BrdUrd. The photolysis-induced DNA strand breaks were, in turn, labeled with digoxygenin- or biotin-conjugated dUTP followed by digoxygenin antibody or avidin, respectively, conjugated with fluorochrome of another color. Alternatively, DNA strand breaks were labeled with BrdUTP which was then detected by FITC-conjugated anti-BrdUrd MoAb. Following counterstaining of cellular DNA with a fluorochrome of a third color it was possible to identify apoptotic cells, cells incorporating BrdUrd, and cells having no DNA strand breaks. Cell fluorescence was measured either by flow cytometry or with the microscope-based laser scanning cytometer. The SBIP approach also offers a possibility to study a colocalization of the immunocytochemically detectable cell constituents at the DNA replication points by microscopy. Using this approach the presence of the proliferating cell nuclear antigen at the DNA replication sites was revealed in MCF-7 breast carcinoma cells.

Antibodies, Monoclonal↗
PubMed1994

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.

Animals↗
PubMed2001

Origins and complexes: the initiation of DNA replication.

Eukaryotic DNA is organized for replication as multiple replicons. DNA synthesis in each replicon is initiated at an origin of replication. In both budding yeast, Saccharomyces cerevisiae and fission yeast, Schizosaccharomyces pombe, origins contain specific sequences that are essential for initiation, although these differ significantly between the two yeasts with those of S. pombe being more complex then those of S. cerevisiae. However, it is not yet clear whether the replication origins of plants contain specific essential sequences or whether origin sites are determined by features of chromatin structure. In all eukaryotes there are several biochemical events that must take place before initiation can occur. These are the marking of the origins by the origin recognition complex (ORC), the loading onto the origins, in a series of steps, of origin activation factors including the MCM proteins, and the initial denaturation of the double helix to form a replication "bubble". Only then can the enzymes that actually initiate replication, primase and DNA polymerase-alpha, gain access to the template. In many cells this complex series of events occurs only once per cell cycle, ensuring that DNA is not re-replicated within one cycle. However, regulated re-replication of DNA within one cell cycle (DNA endoreduplication) is relatively common in plants, indicating that the "once-per-cycle" controls can be overridden.

Cell Cycle↗