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At least 1,783 records · Page 99Linked to original sources
PubMed1999

Initiation of DNA replication in a eukaryotic rolling-circle replicon: identification of multiple DNA-protein complexes at the geminivirus origin.

The mechanism of initiation of DNA replication in eukaryotic rolling- circle replicons is still poorly understood in molecular terms. Geminiviruses, a family of plant DNA viruses, which use this strategy during part of their replicative cycle, replicate in the nucleus and are amenable to molecular studies. Except for the virally encoded initiator protein (Rep), geminivirus DNA replication relies on cellular factors, likely interfering with cell cycle regulation of the infected cell. Here, we report the identification of three distinct DNA-protein complexes of the DNA replication initiator protein encoded by wheat dwarf geminivirus (WDV) within viral regulatory sequences controlling DNA replication and transcription. We have mapped the WDV Rep binding sites by combining gel-shift assays, electron microscopy and DNase I footprinting. Two of the Rep-DNA complexes (C and the V) are high-affinity complexes, located in the proximity of the two divergent TATA boxes, at 150 and 90 bp, respectively, from the DNA replication initiation site. The third one, the O-complex, is a low-affinity complex, which can assemble under conditions supporting the DNA cleavage reaction. This suggests that it might be responsible for initiation of rolling-circle DNA replication in WDV and other members of the Mastrevirus genus.

Base Sequence↗
PubMed1999

Replication control of a small cryptic plasmid of Escherichia coli.

The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.

Bacterial Proteins↗
PubMed2000

Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos.

Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.

Animals↗
PubMed1999

Analysis of the replication elements of the pMJ101 plasmid from the fish pathogen Vibrio ordalii.

Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.

Amino Acid Sequence↗
PubMed2000

Replication of oriJ-based plasmid DNA during the stringent and relaxed responses of Escherichia coli.

The oriJ-based plasmids contain the origin of DNA replication from the cryptic Rac prophage, present in the chromosomes of most Escherichia coli K-12 strains. The organization of the oriJ replication region resembles that of the bacteriophage lambda, although sequence similarity is small. Here we investigated the regulation of replication of the oriJ-based plasmid in E. coli relA(+) and relA(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses. We found that, contrary to plasmids derived from phage lambda, replication of the oriJ-based plasmid proceeds efficiently during both stringent and relaxed responses. On the other hand, density shift experiments and measurement of the stability of a putative replication initiator protein (the lambda O protein homologue) suggest that this replication may be carried out by the heritable replication complex, as previously demonstrated for lambda plasmids. We demonstrate that contrary to bacteriophage lambda p(R) promoter, an analogous promoter from the oriJ region is activated rather than inhibited at increased ppGpp levels. We propose that various responses of these promoters (p(R) and p(R-Rac), which are necessary for transcriptional activation of orilambda and perhaps oriJ, respectively) to ppGpp are responsible for differences in the replication regulation between orilambda- and oriJ-based plasmids during the stringent response.

Artificial Gene Fusion↗
PubMed1994

Functional mapping of regions of the Autographa californica nuclear polyhedrosis viral genome required for DNA replication.

Previous results showed that plasmids containing one of the eight putative origins (ori's) of Autographa californica nuclear polyhedrosis virus (AcMNPV) are replicated after transfection into Spodoptera frugiperda cells if essential trans-acting factors are supplied by AcMNPV infection (Kool et al., Virology, 192, 94-101, 1993a; Kool et al., J. Gen. Virol., in press, 1993b; Leisy and Rohrmann, Virology, 196, 722-730, 1993). In this report a transient complementation assay is described in which four cotransfected cosmid clones, instead of AcMNPV infection, provided essential trans-acting factors for plasmid DNA replication. In this assay plasmid replication was found to be independent of the presence, in cis, of a viral ori. No replication of plasmids occurred when one of the cosmids was omitted from the transfection mixture. This result indicated that this assay is a valid approach for identification of AcMNPV replication genes. We further used the assay to define essential regions in the four required cosmids. Six regions of the AcMNPV genome, EcoRI-I (map unit 0.3-5.8), EcoRI-O (map unit 6.9-8.7), SstI-F (map unit 38.9-45.0), EcoRI-D (map unit 59.9-68.3), a BamHI-SstII fragment of BamHI-B (map unit 84.3-89.7), and EcoRI-B (map unit 90.0-100), with at least seven genes, were found to be essential for plasmid DNA replication. These regions contain the putative DNA polymerase gene (SstI-F), the helicase-like gene (EcoRI-D), and the region where most of the trans-activating immediate--early genes of AcMNPV are located (EcoRI-B). For SstI-F it was shown that this region contains besides the DNA polymerase gene at least one other replication gene. These results show that it will now be possible to define the set of AcMNPV genes necessary and sufficient for DNA replication.

Animals↗
PubMed1994

DNA replication of chimeric JC virus-simian virus 40 genomes.

The ubiquitous virus JCV is the etiologic agent of the human brain disease progressive multifocal leukoencephalopathy. Although infection usually occurs early in life and the virus can remain latent in human tissues, including brain, little information is available regarding its replication. It is known that DNA replication of primate polyomaviruses is dependent upon the synthesis of T antigen and the subsequent interactions of this protein with cellular factors and the viral origin of replication. We constructed chimeric genomes between JCV and SV40, two genetically similar viruses with distinct biologies, in which segments of the T antigen coding region and the replication origin were exchanged. Because the engineering of these genomes created a defect in the structural protein VP1, their DNA replicating activities could be compared without the complication of secondary infection of adjacent cells and amplification of the replication signal. The ability of the JCV-SV40 hybrid T antigens to initiate replication from the two viral origins in primate cells was investigated. A region of the JCV T antigen that includes the DNA binding and zinc finger domains was found to be responsible for the failure of JCV T antigen to interact productively with the SV40 origin. In addition, the ability to replicate in monkey cells was limited to constructs expressing T antigens which contained the carboxy-terminal host range domain of SV40.

Antigens, Polyomavirus Transforming↗
PubMed1995

Identification and characterization of a second putative origin of DNA replication in a baculovirus of Orgyia pseudotsugata.

A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous regions from AcMNPV. In isolation, these repeated sequences were not sufficient for origin activity in OpMNPV-infected Lymantria dispar cells, but required an additional 1.3 kb of sequences located to the left of the repeats. Four regions of the OpMNPV genome that crosshybridize with the repeated region were also found to replicate in our infection-dependent DNA replication assay. A deletion clone of Op5 that replicates efficiently in OpMNPV-infected L. dispar cells, was found to replicate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infected Spodoptera frugiperda cells.

Animals↗
PubMed1999

Site-directed mutagenesis of the AcMNPV p143 gene: effects on baculovirus DNA replication.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a 143-kDa protein (P143) required for viral DNA synthesis and involved in host range determination. The predicted amino acid sequence of P143 contains seven motifs (I, Ia, II-VI) shared with a superfamily of helicases involved in the unwinding of duplex nucleic acids; a putative DNA binding motif; a putative nuclear localization signal (NLS); and a demonstrated host range motif. In this study, the functional significance of these conserved P143 motifs was examined by site-specific mutation resulting in amino acid substitutions of conserved residues within each of them. An in vivo complementation replication assay was developed and each mutated P143 protein expressed from a transfected plasmid was tested for its ability to complement the replication-negative ts8 baculovirus mutant for the amplification of an origin-containing plasmid. Mutations in the helicase motifs I, Ia, and II and in a potential helix-turn-helix motif abolished the ability of P143 to complement the ts8 defect in DNA replication, suggesting that these conserved amino acid residues may be essential for the replication function of the protein. In contrast, mutation of conserved amino acid residues in the helicase motifs IV, V, and VI did not affect the ability of the P143 proteins to complement the replication defect of ts8. A mutation in motif III caused a reduction in the replication function of P143. Deletion of Gly552 in the host range region eliminated the replication function of P143. Mutations within a putative NLS had no effect on the ability of P143 to support DNA replication, suggesting that these residues are nonessential and that the putative P143 NLS sequence may not be responsible for the nuclear localization of the protein. The transient complementation system used in this study provides a simple method for functional analysis of essential baculovirus genes in infected cell cultures.

Adenosine Triphosphate↗
PubMed1975

The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Intermediates of lambda DNA replication in the second half of the latent period after phage lambda infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative forms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as the first round lambda DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the lambda genome. Replicating circles with a linear DNA tail longer than one unit length of lambda genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.

Chromosome Mapping↗
PubMed1989

Transcriptional activation with concurrent or nonconcurrent template replication has differential effects on transient expression from herpes simplex virus promoters.

We have used two methods to induce template replication in order to assess the effect on expression of marker genes controlled by herpes simplex virus type 1 (HSV-1) promoters. One method used the HSV-1 origin of DNA replication from the short repeat region of the viral genome (HSV-1 oris), and allowed simultaneous replication and transcriptional activation of the plasmid-borne template. The other, using the simian virus 40 origin of replication (SV40 ori) allowed plasmid template replication prior to activation of transcription by HSV-1 infection. The two regimes had markedly different effects upon the levels of reporter gene activity induced by HSV-1 superinfection. Replication of reporter plasmids using the SV40 ori yielded levels of reporter gene activity proportional to plasmid copy number when cells were superinfected with HSV-1. In contrast, our results indicated that sequences containing, or in close proximity to, the HSV-1 oris in the reporter plasmid had a significant inhibitory effect on expression from all viral promoters whether or not the plasmid was allowed to replicate. Still, the early (beta) promoter-controlled reporter enzyme activity declined at late times while that controlled by the strict late (gamma) promoter was significantly higher following HSV-1 oris-mediated template replication.

Animals↗
PubMed1996

Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28.

A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication. In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified. The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes. The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A. eutrophus. The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E. coli phage. The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28.

Alcaligenes↗
PubMed2000

The effects of transcription and RNA processing on the initiation of chloroplast DNA replication in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the origin for chloroplast DNA replication, Ori A, overlaps the coding region for the chloroplast ribosomal protein Rpl16. In an in vitro DNA replication system that uses cloned Ori A as template, alteration of transcription across rpl16 affects replication activity. S1 nuclease protection mapping of cellular RNA derived from this region revealed multiple 5' and 3' ends, and several 3' ends were mapped within mini Ori A (224 bp), the core region for replication initiation. We also demonstrated that the protein fraction used in the in vitro DNA replication system contained an RNA processing activity responsible for the generation of multiple 3' ends. The 3' ends of some of the processed RNA species coincided with those of the cellular transcripts. Initiation of DNA replication in the in vitro system changed the abundance of some of the processed RNA species, and the S1 nuclease protection pattern generated by the 3' ends now mimicked that of the in vivo transcripts. We also monitored the pattern of 3' ends in cellular transcripts from the rpl16 region during gametogenesis--when the chloroplast DNA is under-replicated--and detected a change in transcript abundance that correlated with that seen in the in vitro study. Measurements of the template activity of mutants with targeted sequences change near the sites of processing also supported the notion that the processed transcripts play an important role in DNA replication.

Animals↗
PubMed1984

Fidelity of DNA replication under conditions used for oligodeoxynucleotide-directed mutagenesis.

The fidelity of DNA replication in vitro by DNA polymerase I (large subfragment) of Escherichia coli has been measured by the standard bioassay: single-stranded phi X174 DNA (plus strand) containing an amber codon was primed with a synthetic oligodeoxynucleotide, replicated and the frequency of point mutations formed in the synthetic minus strand of the resultant double-stranded DNA determined from the number of revertant phage produced in a spheroplast assay. Since the assay depends crucially on the frequency of expression of the mutations in the heteroduplex, and this can vary for a variety of reasons, parallel control experiments were performed using a primer that covered the amber codon but contained the same mismatch that occurred during replication. The frequency of expression of these mutations was found to vary from 40 to 100% in fully ligated heteroduplexes, depending upon the age and batch of spheroplasts used. The variation probably reflects the viability of the post-replicative mismatch repair enzymes in the spheroplasts used for transfection. Far lower frequencies of expression were found under conditions of poor replication. Accurate data and rate laws for fidelity are obtained only when the bioassay is normalized for the variation in the expression frequency. There is active proofreading by the 3'-5'-exonuclease activity of the polymerase of a misincorporation resulting from a dGTP:T mismatch. The contribution of proofreading to fidelity is low: accuracy is enhanced by a factor of less than 7 at the concentrations of dNTPs in vivo. The lower accuracy of Pol I than Pol III is due mainly to poorer proofreading, which is manifested in a lower "cost" of replication: only 0.7 to 1.7% of the dNTPs are turned over to dNMPs during replication compared with 6 to 13% for Pol III. The error rates measured for Pol I under conditions used for oligodeoxynucleotide-directed mutagenesis are sufficiently low that extraneous errors should not be induced when the concentrations of dNTPs are balanced. However, even higher fidelity will be obtained using the lowest concentrations of dNTPs consistent with efficient replication (approximately 20 microM). Highly unbalanced concentrations as used in pulsed labelling should be avoided.

Bacteriophage phi X 174↗
PubMed1985

Bacteriophage P4 DNA replication. Location of the P4 origin.

An electron microscopic examination of replicating bacteriophage P4 DNA molecules has revealed theta-type structures that replicate bidirectionally from a single origin. Many replicating P4 DNA molecules also contain long (2000 bases) single-strand DNA regions at the growing fork that are deployed in a trans configuration, which supports the concept of continuous leading strand and discontinuous lagging strand syntheses. The position of the P4 origin was localized by the use of a plasmid complementation test for replication in vivo, as well as by labeling of DNA replicating in vitro in the presence of a chain-terminating inhibitor. During this study we discovered a second site on the P4 genome which is essential for replication, and we have named it crr (cis region required for replication). The site is located at least 3300 bases from the origin but appears to be required for the initiation of DNA replication in vivo as well as in vitro.

Coliphages↗
PubMed1991

Factors contributing to the restricted DNA replicating activity of JC virus.

The basis for the restricted host range behavior of JC virus (JCV) in vitro was investigated by focusing on its DNA replicating activity and comparing it to that of simian virus 40 (SV40). Prototype, mutant, and hybrid JCV and SV40 DNAs were tested for their replicating activity in cells permissive for one or both of the viruses. Results from these experiments indicated that, relative to its SV40 counterpart, the JCV T antigen functioned less efficiently and was more specific in its interactions with polyomavirus DNA replication origins. The JCV T antigen exhibited a lower specific DNA binding activity than did the SV40 T antigen, which might contribute to this virus' reduced DNA replicating activity. However, the JCV protein did bind to both the JCV and SV40 replication origins with similar efficiency, indicating that the ability of the JCV T antigen to discriminate between the JCV and SV40 origins involved a step subsequent to specific DNA binding. The results also suggested that the failure of JCV to replicate to detectable levels in monkey kidney cells was due to the inefficient interactions of its T protein with the viral origin and the host replication machinery. The inability of the JCV T antigen to carry out one or more of these DNA replication functions efficiently contributes to the restricted lytic behavior of this virus.

Animals↗
PubMed1979

The asymmetric segregation of parental nucleosomes during chrosome replication.

SV40 DNA replicated in the presence of cycloheximide was more sensitive to staphylococcal nuclease digestion and had a lower superhelical density than viral DNA replicated in the absence of this drug. These data indicate that fewer nucleosomes are associated with progeny SV40 DNA molecules after DNA replication in the absence of protein synthesis and that these nucleosomes are derived from the parental histones. We designed an experiment to determine whether these parental SV40 nucleosomes segregate to the leading side of the replication form where DNA synthesis is continuous, the lagging side of the fork where DNA synthesis is discontinuous or randomly to both sides of the fork. The results indicate that the parental histones distributed themselves asymmetrically, preferentially (80-90%) segregating with the leading side of both SV40 DNA replication forks during bidirectional replication in the absence of protein synthesis. In the case of SV40, the same parental DNA strands are the templates for the leading side of DNA replication at both forks as well as the templates for the informational or coding strand of early and late viral mRNA synthesis. Based on this correspondence, we designed an experiment to test whether chicken cells growing in culture and replicating their DNA in the absence of protein synthesis segregated their parental histones asymmetrically to the progeny DNA strand that also coded for stable nuclear RNA transcripts. The results of these experiments indicate that, like SV40, parental cellular histones segregate asymmetrically and are preferentially associated with those DNA template strands that code for stable nuclear RNA species detected by hybridization to single-copy DNA.

Cycloheximide↗
PubMed1995

ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome.

The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

Cell Cycle Proteins↗