SEARCH · PubMed Health

Results for “Replicability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed1999

Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast.

In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320. Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

Base Sequence↗

PubMed2006

Sap1 promotes the association of the replication fork protection complex with chromatin and is involved in the replication checkpoint in Schizosaccharomyces pombe.

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.

Chromatin↗

PubMed1994

Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Base Sequence↗

PubMed1998

Replication protein A as a potential regulator of DNA replication in cells exposed to hyperthermia.

It is well known that exposure of cells to heat leads to a drastic inhibition of DNA synthesis as assayed in vivo by the incorporation of radioactive precursors into acid-insoluble material. Here we introduce an SV40 in vitro DNA replication assay and show that this inhibition may be partly due to the activation of a checkpoint in S phase that stalls the initiation of DNA replication by inactivating replication protein A (RPA), an essential factor for replication. The results implicate trans-acting processes in the regulation of DNA replication after heat exposure and suggest that such processes may be an integral part of the normal response to heat insult. The observations extend and complement previous studies that have implicated heat-induced chromatin damage acting in cis as a cause for the observed inhibition of DNA synthesis in cells exposed to hyperthermia. A model is proposed postulating that the presence of single-stranded DNA, or heat-induced damage to chromatin structures directly, albeit passively, inhibits the elongation stages of ongoing DNA replication. It is hypothesized that arrested replication forks subsequently act as signals to activate the S-phase checkpoint that actively inhibits the initiation of new replicons. The ultimate purpose of this response will be the minimization of the toxic consequences of heat-induced damage, as it may delay DNA replication until chromatin conformation has been restored. DNA replication in the presence of chromatin damage has been implicated in the formation of lethal chromosome aberrations observed in cells heated during S phase. The operation of active processes in the regulation of DNA replication in cells exposed to hyperthermia offers new targets for intervention and sensitization of cells to heat.

Cytoplasm↗

PubMed2002

DNA replication joins the revolution: whole-genome views of DNA replication in budding yeast.

Replication origins, which are responsible for initiating the replication of eukaryotic chromosomal DNAs, are spaced at intervals of 40 to 200 kb. Although the sets of proteins that assemble at replication origins during G(1) to form pre-replicative complexes are highly conserved, the structures of replication origins varies from organism to organism. The identification of replication origins has been a labor-intensive task, requiring the analysis of chromosomal DNA replication intermediates. As a result, only a few replication origins have been identified and studied. In a pair of recently published papers, Raghuraman and colleagues and Wyrick, Aparicio and colleagues provide complementary microarray-based approaches to the identification of replication origins. These genome-wide views of DNA replication in Saccharomyces cerevisiae provide new insights into the way that the genome is duplicated and hold promise for the analysis of other genomes.

Biological Evolution↗

PubMed1993

Cell fusion-induced quick change in replication time of the inactive mouse X chromosome: an implication for the maintenance mechanism of late replication.

It is unknown how and why the genetically inactivated mammalian X chromosome replicates late in S phase. There are also occasional inactive X chromosomes characterized by an opposite behavior replicating early in S phase. Two clonal cell lines, MTLB3 and MTLH8, isolated from a cultured murine T-cell lymphoma have an allocyclic X chromosome of the latter type. This precociously replicating X chromosome was judged to be genetically inactive as the late replicating one. Immediately after fusion with another cell line, the precociously replicating X chromosome from these cells starts to replicate late in S phase. This finding seems to suggest that late replication characterizing the inactive X chromosome is actively maintained by a trans-acting factor in female somatic cells, and that its lack entails a switch from late replication to precocious replication. It remains unknown whether this presumptive factor also modifies the autosomal replication pattern.

Animals↗

PubMed1979

Induction of UV-resistant DNA replication in Escherichia coli: induced stable DNA replication as an SOS function.

The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA+ lexA+-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with malidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvrA6 strain and a uvr+ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.

Bacterial Proteins↗

PubMed1991

Classes of autonomously replicating sequences are found among early-replicating monkey DNA.

Thirteen new independent clones of origin-enriched sequences (ors) that are capable of autonomous replication have been identified from a library of 100 ors i clones that had been previously isolated from early replicating monkey (CV-1) DNA. Autonomous replication was assayed by transient episomal replication in transfected HeLa cells; ors-plasmid DNA was isolated at various times after transfection and screened by the DpnI resistance assay and the bromodeoxyuridine (BrdUrd) substitution assay to differentiate between input and newly replicated DNA. Four of the autonomously replicating clones were identified by screening the ors-library with probes of ors 3, 8, 9 and 12, previously shown to be capable of autonomous replication (Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA (1987) 84, 6668-6672). The other nine functional ors clones were identified among 18 randomly chosen ones, which were similarly screened for autonomous replication. Nucleotide sequence analyses of 11 of the newly identified functional ors plasmids revealed, in most of them, features similar to those present in other viral or eukaryotic replication origins, notably the presence of AT-rich regions and inverted repeats. Pairwise comparisons between the newly identified ors showed no extensive sequence homologies, other than the presence of the alpha-satellite repetitive sequence family in three ors and of the repetitive Alu sequence family in one ors. The results suggest that there exist different classes of mammalian replication origin, highly or moderately repetitive and unique, and that their activation is most probably dependent on the presence of structural determinants rather than on a particular sequence.

Animals↗

PubMed1994

Loading of a DNA helicase on the DNA unwinding element in the yeast replication origin: mechanism of DNA replication in a model system.

We found that initiation of DNA replication occurs from the region containing the yeast autonomously replicating sequence 1 (ARS1), by incubating negatively supercoiled plasmid DNA with the proteins required for SV40 DNA replication in addition to DNA gyrase (Ishimi, Y., & Matsumoto, K. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5399-5403). Here, the mechanism of DNA replication and the roles of the replication proteins in this model system were analyzed. Both SV40 T antigen as a DNA helicase and multisubunit human single-stranded DNA binding protein (HSSB) (also called RP-A) were required for the initial step of DNA synthesis. Furthermore, it has been shown that T antigen plays an essential role in the initiation of DNA replication from the ARS region in this system. The digestion of negatively supercoiled DNA with the single-strand-specific nuclease P1 revealed that regions containing A, B, and C domains of ARS1 can be unwound under the conditions used for DNA replication. Footprinting with KMnO4 indicated that T antigen interacted with the unwound B domain where initiation of DNA replication mainly occurred. When circular DNAs of different negative-superhelical densities were replicated in the absence of DNA gyrase, short fragments were synthesized from the ARS region in proportion to its density and they were elongated by addition of HeLa topoisomerase I, which inhibits the initiation of DNA replication in this system.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Polyomavirus Transforming↗

PubMed1996

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

3T3 Cells↗

PubMed2002

Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing.

Replication of the genome before mitotic cell division is a highly regulated process that ensures the fidelity of DNA duplication. DNA replication initiates at specific locations, termed origins of replication, and progresses in a defined temporal order during the S phase of the cell cycle. The relationship between replication timing and gene expression has been the subject of some speculation. A recent genome-wide analysis in Saccharomyces cerevisiae showed no association between replication timing and gene expression. In higher eukaryotes, the limited number of genomic loci analyzed has not permitted a firm conclusion regarding this association. To explore the relationship between DNA replication and gene expression in higher eukaryotes, we developed a strategy to measure the timing of DNA replication for thousands of genes in a single DNA array hybridization experiment. Using this approach, we generated a genome-wide map of replication timing for Drosophila melanogaster. Moreover, by surveying over 40% of all D. melanogaster genes, we found a strong correlation between DNA replication early in S phase and transcriptional activity. As this correlation does not exist in S. cerevisiae, this interplay between DNA replication and transcription may be a unique characteristic of higher eukaryotes.

Animals↗

PubMed1996

Vaccinia virus DNA replication: two hundred base pairs of telomeric sequence confer optimal replication efficiency on minichromosome templates.

Vaccinia virus is a complex DNA virus that exhibits significant genetic and physical autonomy from the host cell. Most if not all of the functions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in identifying trans-acting factors involved in DNA synthesis, the mechanism of genome replication has remained poorly understood. The genome is a linear duplex with covalently closed hairpin termini, and it has been presumed that sequences and/or structures within these termini are important for the initiation of genome replication. In this report we describe the construction of minichromosomes containing a central plasmid insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichromosomes was compared with a control substrate containing synthetic hairpin termini, specificity for viral telomeres was apparent. Inclusion of > or = 200 bp from the viral telomere was sufficient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective. Chimeric 200-bp telomeres containing the 65-bp terminal element and 135 bp of ectopic sequence also failed to confer efficient replication, providing additional evidence that telomere function is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coincident with viral replication, and generated covalently closed minichromosome products. These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected cells.

Animals↗

PubMed1997

Replication synchrony-PCR: a sampling-time-independent assay for replication synchrony in human tissues and tumors in situ.

Replication synchrony within a cell population can be demonstrated by pulse-labeling followed by PCR amplification of immunoprecipitated 5-iodo-2'-deoxyuridine (IdUrd)-labeled DNA from cells of otherwise indeterminant kinetic stages. This replication synchrony-PCR approach may be valuable in understanding the dynamics of human normal tissue or solid tumor replication in situ where access for repeated sampling is severely limited. IdUrd labeling provides a sampling-time-independent method for assessing the replicative status of a cell population at the time when the label was presented. Using genes whose time of replication in S phase is already known, the presence of a cell in early or late S phase can be determined and a qualitative measure made of replication synchrony in the population. This approach was evaluated in synchronous and random cultures of Ej cells using the early replicating PGK-1 gene to identify cells in early S phase at the time of labeling and the late replicating factor IX gene to identify cells that were in late S phase. To test the feasibility of clinical application of this technique, human tumor cells from patients with advanced cancers, given IdUrd therapeutically at specified times of the day, were evaluated. In some patients, replication synchrony-PCR provided evidence of parasynchronous DNA replication in tumor cells. This technique could be appended to existing clinical studies in which BrdUrd or IdUrd is being given to patients either diagnostically or therapeutically.

Cell Division↗

PubMed1997

A variable domain of delayed replication in FRAXA fragile X chromosomes: X inactivation-like spread of late replication.

The timing of DNA replication in the Xq27 portion of the human X chromosome was studied in cells derived from normal and fragile X males to further characterize the replication delay on fragile X chromosomes. By examining a number of sequence-tagged sites (STSs) that span several megabases of Xq27, we found this portion of the normal active X chromosome to be composed of two large zones with different replication times in fibroblasts, lymphocytes, and lymphoblastoid cells. The centromere-proximal zone replicates very late in S, whereas the distal zone normally replicates somewhat earlier and contains FMR1, the gene responsible for fragile X syndrome when mutated. Our analysis of the region of delayed replication in fragile X cells indicates that it extends at least 400 kb 5' of FMR1 and appears to merge with the normal zone of very late replication in proximal Xq27. The distal border of delayed replication varies among different fragile X males, thereby defining three replicon-sized domains that can be affected in fragile X syndrome. The distal boundary of the largest region of delayed replication is located between 350 and 600 kb 3' of FMR1. This example of variable spreading of late replication into multiple replicons in fragile X provides a model for the spread of inactivation associated with position-effect variegation or X chromosome inactivation.

Cell Cycle↗

PubMed1995

Replication error rates for G.dGTP, T.dGTP, and A.dGTP mispairs and evidence for differential proofreading by leading and lagging strand DNA replication complexes in human cells.

We have determined the fidelity of DNA replication by human cell extracts in reactions containing excess dGTP. Replication errors were scored using two M13 DNA substrates having the replication origin on opposite sides of the lacZ alpha-complementation gene. The data suggest that the average rates for replication errors resulting from G(template), T.dGTP, and A.dGTP mispairs are 25 x 10(-6), 12 x 10(-6), and 3 x 10(-6), respectively. The data also suggest that error rates for both the (+) and (-) strands differ by less than 2-fold when they are replicated either as the leading or lagging strand. This is in contrast to the 33- and 8-fold differences observed earlier for G.dTTP and C.dTTP mispairs on the (+) strand when replicated by the leading or lagging strand complex (Roberts, J. D., Izuta, S., Thomas, D. C., and Kunkel, T. A. (1994) J. Biol. Chem. 269, 1711-1717). Thus, the relative fidelity of the leading and lagging strand replication proteins varies with the mispair and sequence considered. Misincorporation of dGTP preferentially occurs at template positions where dGTP is the next correct nucleotide to be incorporated. This "next nucleotide" effect is characteristic of reduced exonucleolytic proofreading and suggests that these replication errors are normally proofread efficiently. Fidelity measurements performed in the absence or presence of dGMP, an inhibitor of proofreading exonuclease activity, suggest that the leading strand replication complex proofreads some mispairs more efficiently than does the lagging strand replication complex.

Base Sequence↗

PubMedDate not supplied

Control of DNA replication: regulation and activation of eukaryotic replicative helicase, MCM.

DNA replication is a key event of cell proliferation and the final target of signal transduction induced by growth factor stimulation. It is also strictly regulated during the ongoing cell cycle so that it occurs only once during S phase and that all the genetic materials are faithfully duplicated. DNA replication may be arrested or temporally inhibited due to a varieties of internal and external causes. Cells have developed intricate mechanisms to cope with the arrested replication forks to minimize the adversary effect on the stable maintenance of genetic materials. Helicases play a central role in DNA replication. In eukaryotes, MCM (minichromosome maintenance) protein complex plays essential roles as a replicative helicase. MCM4-6-7 complex possesses intrinsic DNA helicase activity which translocates on single-stranded DNA form 3' to 5'. Mammalian MCM4-6-7 helicase and ATPase activities are specifically stimulated by the presence of thymine-rich single-stranded DNA sequences onto which it is loaded. The activation appears to depend on the thymine content of this single-strand, and sequences derived from human replication origins can serve as potent activators of the MCM helicase. MCM is a prime target of Cdc7 kinase, known to be essential for activation of replication origins. We will discuss how the MCM may be activated at the replication origins by template DNA, phosphorylation, and interaction with other replicative proteins, and will present a model of how activation of MCM helicase by specific sequences may contribute to selection of replication initiation sites in higher eukaryotes.

Animals↗

PubMed1992

Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication.

Chinese hamster genomic DNA sequences from the region downstream of the dihydrofolate reductase (DHFR) gene reported to contain a chromosomal origin of bidirectional DNA replication (OBR-1) were tested for their ability to support autonomous DNA replication in human cells. A 13.3 kilobase fragment containing OBR-1 and surrounding sequences supported replication in short-term and long-term replication assays, while a 4.5 kb fragment containing OBR-1 did not support substantial replication in either assay. These results are consistent with our previous observations that large fragments of human DNA support replication, while smaller fragments are less efficient. The replication activities of plasmids containing OBR-1 were no greater than those of randomly chosen human fragments of similar size. Furthermore, two-dimensional gel analysis of plasmids containing OBR-1 indicated that initiation does not preferentially occur within the OBR-1 region. These results suggest that in the context of autonomous replication, the DHFR sequences tested do not contain genetic information specifying site-specific replication initiation. Possible implications of these results for chromosomal replication are discussed.

Animals↗

PubMed2002

Directionality of lambda plasmid DNA replication carried out by the heritable replication complex.

There are two 'pathways' of replication of lambda plasmids in Escherichia coli. One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round. Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences. Here we investigated directionality of lambda plasmid replication carried out by the heritable and newly assembled replication complexes. Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions. The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.

Bacteriophage lambda↗