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At least 289 records · Page 16Linked to original sources
PubMed1992

Transcription in vivo within the replication origin of the Escherichia coli chromosome: a mechanism for activating initiation of replication.

Within the replication origin, oriC, of the Escherichia coli chromosome, novel in vivo transcripts were detected which proceeded rightward and whose production was activated by DnaA protein. In contrast, DnaA protein repressed the previously described ori-L leftward transcription. The former should introduce negative supercoiling, and the latter positive supercoiling, into the 13-mers. The effects of transcription on the initiation of replication were also investigated by making constructs with promoters placed near oriC. Transcription was found to enhance the origin activity only when it was oriented in such a way as to introduce negative supercoiling into the 13-mers. From these results, we propose that transcription within oriC regulates replication initiation by altering the topology of the 13-mer region.

Base Sequence↗
PubMed2000

Premature structural changes at replication origins in a yeast minichromosome maintenance (MCM) mutant.

The Cdc7p protein kinase in the budding yeast Saccharomyces cerevisiae is thought to help trigger DNA replication by modifying one or more of the factors that assemble at replication origins (ARSs). To investigate events catalyzed by Cdc7p, we compared the structure of replication origins in cells containing conditional mutations in Cdc7p and Cdc8p, a thymidylate kinase that is required for DNA synthesis. High resolution genomic footprinting indicated that the presumptive lagging strand template in ARS1 became highly sensitive to KMnO(4) modification after the CDC7 execution point. These results suggested that Cdc7p triggers DNA unwinding. The transition from late G(1) phase to the CDC7 execution point and from the CDC7 to the CDC8 execution points was accompanied by small but ARS-dependent changes in DNA topology. These results suggested that DNA unwinding before the CDC8 execution point either is highly localized or that the torsional stress associated with initial DNA unwinding is minimized by compensatory protein-DNA structural changes. The ARS DNA structural attributes evident in cells blocked at the CDC8 execution point were also evident in alpha-factor-blocked, G(1) phase cells containing the CDC7 bypass mutant mcm5/cdc46-bob1. This result strongly suggests that the structural changes during the transition from the CDC7 to CDC8 execution points depend on the Cdc7p protein kinase and involve alteration of the minichromosome maintenance protein complex.

Base Sequence↗
PubMed2006

Binding of the universal minicircle sequence binding protein at the kinetoplast DNA replication origin.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.

Animals↗
PubMed1982

Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli.

The chromosomal DNA replication origins (oriC) from two members of the family Enterobacteriaceae, Enterobacter aerogenes and Klebsiella pneumoniae, have been isolated as functional replication origins in Escherichia coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system (Fuller, et al., Proc. Natl. Acad. Sci. U.S.A. 78:7370--7374, 1981), and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. We show that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.

Bacterial Proteins↗
PubMed1984

Random distribution of mammalian replication origins in matrix and total nuclear DNA.

Nuclear matrices from mouse and rat tumour cells were isolated and characterized by their microscopic appearance, protein profiles and DNA content. They presented well-defined structures containing 15-20% of the nuclear protein and 1-3% of the nuclear DNA. Matrix DNAs were immobilized on nitrocellulose filters and hybridized to nick-translation 32P-labelled homologous DNA fragments containing the corresponding replication origins. As control total nuclear DNAs were also immobilized on filters and hybridized to origin-containing DNAs. The origin-containing DNAs hybridized to the same extent to both matrix and total DNAs, which showed that they contained the same proportion of origin sequences. In an alternative series of experiments, plasmids containing either rat or mouse replication origins were immobilized on filters and were hybridized with in vitro 32P-labelled matrix and total nuclear DNAs. Here again both matrix and total nuclear DNAs hybridized to the same extent with the origin-carrying plasmids, which showed that neither rat nor mouse matrix DNAs were enriched in DNA replication origin sequences.

Animals↗
PubMed1980

Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication.

A composite plasmid (pXX11) was constructed by joining of an oriC plasmid (pMCR115) carrying the replication origin (oriC) of the Escherichia coli chromosome and a mini-F plasmid (pSC138) carrying the ampicillin-resistance gene (bla). Plasmid pXX11 can replicate, by using oriC, in Hfr cells and mafA mutant cells that cannot support replication of an F plasmid. This plasmid is stably maintained in these host cells during cell growth even under nonselective conditions by use of the partition mechanism of the mini-F genome. In contrast to other oriC plasmids reported previously, pXX11 has no detectable effect on host cell growth. Higher copy-number (Cop-) mutants of pXX11 were isolated, and some of them were found to carry an insertion or deletion within a region derived from the E. coli chromosome. This region, designated cop (copy number), covers about 0.7 kilobase pair and is located approximately 3 kilobase pairs away from the oriC region at the side opposite the asn gene. Evidence suggests that the normal cop region locted on the oriC plasmid acts to reduce the copy number of the plasmid. Plasmid pXX11 complements the uncB402 mutation located on the host chromosome, but some of the Cop- plasmids do not, suggesting that the cop region is vey closely linked to uncB.

DNA Replication↗
PubMed1999

Selection and mapping of replication origins from a 500-kb region of the human X chromosome and their relationship to gene expression.

In higher eukaryotes the mechanism controlling initiation of DNA replication remains largely unknown. New technologies are needed to shed light on how DNA replication initiates along the genome in specific regions. To identify the human DNA sequence requirements for initiation of replication, we developed a new method that allows selection of replication origins starting from large genomic regions of human DNA. We repeatedly isolated 15 new putative replication origins (PROs) from a human DNA region of 500 kb in which 17 genes have previously been characterized. Fine-mapping of these PROs showed that DNA replication can initiate at many specific points along actively transcribed DNA in the cell lines used for our selection. In conclusion, in this paper we describe a new method to identify PROs that suggests that the availability of initiation sites is dependent on the transcriptional state of the DNA.

Blotting, Southern↗
PubMed1997

Mapping replication origins by nascent DNA strand length.

The mapping of replication origins by nascent DNA strand length determination is a very sensitive generally applicable method that identifies even single-copy origins in mammalian chromosomes. A major advantage of this procedure is that there is no need for synchronization of cells or treatment with metabolic agents, which allows the origin to be studied under physiological conditions. This technique is based upon the amplification of specific sequence markers on nascent DNA strands that initiated replication within the region of the putative origin. Therefore, this method requires detailed sequence information of the locus to be analyzed. As a first step, nascent DNA of proliferating cells is pulse-labeled with BrdU followed by size fractionation and purification with anti-BrdU antibodies. The position of putative origins can then be determined via identification of the shortest nascent strands that can be amplified by PCR and hybridized to probes homologous to the amplified segments. Here, we give a detailed description of the theory behind the method and a full recipe for its application. Advantages and limitations of the procedure are discussed.

Animals↗
PubMed2001

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing.

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Alkaloids↗
PubMed1996

A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli.

We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC. Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo.

Bacterial Proteins↗
PubMed1982

Replication origins are associated with transcription initiation sequences in the mitochondrial genome of yeast.

Mitochondrial transcripts have been investigated in a series of spontaneous petite mutants of Saccharomyces cerevisiae endowed with mitochondrial genomes formed by short repeat units containing no genes, but either: (a) one of the seven ori sequences, the canonical origins of DNA replication (ori+ mutants); or (b) partially deleted ori sequences, lacking GC-rich clusters A or C (ori- mutants); or (c) no canonical ori sequence, but only oris sequences, the surrogate origins of replication ( orio mutants). The results indicate that some ori sequences play a role in transcription initiation, and that the presence of cluster C and, more specifically, of an AT-rich sequence next to it, are essential for transcription to take place. Hybridization experiments with separated DNA strands have identified the template strand used in transcription as the strand containing the oligopyrimidine stretch of cluster C. S1 degradation of RNA-DNA hybrids indicated that transcription initiates at a TATTACTTATATATTT sequence next to the oligopyrimidine stretch of cluster C and proceeds in the cluster C----cluster A direction. The relevance of these results for the transcription of the wild-type mitochondrial genome is discussed.

Base Sequence↗
PubMed2006

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Alloxan↗
PubMed1988

Functional analysis of the leading strand replication origin of plasmid pUB110 in Bacillus subtilis.

Supercoiled plasmid DNA is the substrate for initiation of pUB110 replication, and - by inference - for binding of its initiator protein (RepU) to the plasmid replication origin (oriU) in vivo. No hairpin structure is required for RepU-oriU recognition. RepH (the pC194 replication initiation protein) failed to initiate replication in trans at oriU. The nucleotides that determine the specificity of the replication initiation process are located within oriU but termination is unefficient. Therefore the segment that forms the full recognition signal for termination is probably located 3' of the oriU recognition sequence. Two overlapping domains, one for initiation and one required for termination, compose the leading strand replication origin of plasmid pUB110.

Bacillus subtilis↗
PubMed1988

AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC.

The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system. This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping. From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level. gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control. However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA. The gidA promoter was mapped by deletion analysis and by S1 mapping. We defined two regions that affect promoter activity negatively. Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region. These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin.

Asparaginase↗
PubMed1992

Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe.

In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.

Chromosomes, Fungal↗
PubMed1981

Replication of M13 oriC bacteriophages in Escherichia coli rep mutant is dependent on the cloned Escherichia coli replication origin.

The involvement of the Escherichia coli rep protein in the replication of M13 chimeric deoxyribonucleic acids (DNAs) carrying the E. coli chromosomal DNA replication origin (oriC) has been examined. Previous studies indicate that the cloning of a 3,550-base-pair sequence of chromosomal DNA containing oriC into an M13 vector allows extensive replication of the M13 oriC chimeric DNA in an E. coli rep-3 mutant. We have extended these studies by preparing a 330-base-pair deletion that specifically deletes the oriC sequence in the M13 oriC DNAs, to demonstrate that the replication observed in the rep-3 host is dependent on the cloned origin. Thus, a DNA-unwinding enzyme other than the rep protein may be involved in the strand separation process accompanying replication which initiates at oriC in the M13 oriC chimeric DNAs and in the E. coli chromosome. The rep assay used for assessing the functionality of the cloned oriC is useful for analysis of any rep-independent origin of replication functional in E. coli. A direct selection for a cloned origin of replication is possible in the rep-3 recA56 host. Since the cloned origin is nonessential for propagation of the M13 chimeric phage in a rep+ host, mutations in the cloned origin may be constructed, and the mutant phage may be examined by a simple transductional analysis of the rep-3 recA56 mutant strain.

Coliphages↗
PubMed1991

Adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin.

The adeno-associated virus (AAV) rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of the rep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the carboxyl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin.

Amino Acid Sequence↗
PubMed2026

Ori-Finder-Arch: An Updated Web Server for the Annotation and Visualization of Archaeal Replication Origins.

Archaea are promising chassis organisms in biotechnology, and the accurate annotation of their chromosomal replication origins (oriCs) is the key to unlocking their full potential. However, the existing Ori-Finder 2 web server suffers from low accuracy, slow speed, and limited scalability. In this study, we present Ori-Finder-Arch, an updated web server for high-performance oriC prediction in archaea. This pipeline integrates HMMER-based replication initiation protein (RIP) annotation, refined consensus motif recognition, and GC profile-based DNA unwinding element (DUE) detection. On a benchmark set of experimentally validated oriCs, Ori-Finder-Arch achieved a recall of 95.6% and a precision of 86.0%, substantially outperforming Ori-Finder 2 (62.2% and 63.6%, respectively), while running 4.75 times faster and supporting diverse assembly levels. When applied to the available archaeal assemblies, it successfully annotated 17,472 oriCs. Meanwhile, the web server provides interactive visualizations at different levels. In conclusion, Ori-Finder-Arch offers an efficient, accurate, and user-friendly platform for advanced studies of archaeal DNA replication initiation and synthetic biology applications, and is freely available at https://tubic.org/Ori-Finder-Arch/ and https://tubic.tju.edu.cn/Ori-Finder-Arch/.

Archaea↗