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At least 397 records · Page 22Linked to original sources
PubMed1998

Cell cycle modulation of protein-DNA interactions at a human replication origin.

We followed the variations of protein-DNA interactions occurring in vivo over the early firing replication origin located near the human lamin B2 gene, in IMR-90 cells synchronized in different moments of the cell cycle. In G0 phase cells no protection is present; as the cells progress in G1 phase an extended footprint covering over 100 bp appears, particularly marked at the G1/S border. As the cells enter S phase the protection shrinks to 70 bp and remains unchanged throughout this phase. In mitosis the protection totally disappears, only to reappear in its extended form as the cells move into the next G1. These variations are reminiscent of those corresponding to the formation of the pre- and post-replicative complexes described in yeast and Xenopus cells.

Cell Cycle↗
PubMed1989

The DNA unwinding element: a novel, cis-acting component that facilitates opening of the Escherichia coli replication origin.

We have discovered that DNA supercoiling, in the absence of replication proteins, induces localized unwinding in the Escherichia coli replication origin (oriC) at the same sequence opened by the dnaA initiator protein. The DNA helix at the tandemly repeated, 13mer sequence is thermodynamically unstable, as evidenced by hypersensitivity to single-strand-specific nuclease in a negatively supercoiled plasmid, and demonstrated by stable DNA unwinding seen after two-dimensional gel electrophoresis of topoisomers. A replication-defective oriC mutant lacking the leftmost 13mer shows no nuclease hypersensitivity in two remaining 13mers and no detectable DNA unwinding on two-dimensional gels. The replication defect in the oriC mutant can be corrected by inserting a dissimilar DNA sequence with reduced helical stability in place of the leftmost 13mer. Thus, the helical instability of the leftmost 13mer, not the specific 13mer sequence, is essential for origin function. The rightmost 13mer exhibits helical instability but differs from the leftmost 13mer in its strict sequence conservation among related bacterial origins. The repeated 13mer region appears to serve two overlapping functions: protein recognition and helical instability. We propose that the cis-acting sequence whose helical instability is required for origin function be called the DNA unwinding element (DUE).

Base Sequence↗
PubMed1998

Replication origins in yeast versus metazoa: separation of the haves and the have nots.

The recent flood of information concerning Saccharomyces cerevisiae replication origins and the proteins that interact with them contrasts alarmingly to the trickle of progress in our understanding of metazoan origins. In mammalian cells, origins are complex and heterogeneous, and appear to be selected by features of nuclear architecture that are re-established after each mitosis. Studies in Xenopus egg extracts have shown that once per cell cycle replication does not require specific origin sequences, despite the identification of functional homologues to yeast origin-binding proteins. These observations suggest that initiation of DNA replication in higher eukaryotes is focused to specific genomic regions by features of chromosome structure.

Animals↗
PubMed2001

Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins.

BACKGROUND: In order to ensure precise chromosome duplication, eukaryotes "license" their replication origins during late mitosis and early G1 by assembling complexes of Mcm2-7 onto them. Mcm2-7 are essential for DNA replication, but are displaced from origins as they initiate, thus ensuring that no origin fires more than once in a single cell cycle. RESULTS: Here we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occurring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-gamma-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-gamma-S. CONCLUSIONS: Nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.

Animals↗
PubMed1985

The nucleotide sequences of the replication origins of plasmids ColA and ColD.

The nucleotide sequences of the replication origin regions of plasmids ColA-CA31 and ColD-CA23 were obtained. Analysis of the nucleotide sequences showed a high degree of homology of these regions with the plasmid ColE1 region responsible for its autonomous replication. In the ColA and ColD regions involved in the regulation of replication, sites have been revealed identical to those participating in the transcription initiation of the ColE1 plasmid RNA I and primer RNA. The presumed RNA polymerase binding sites and the RNA polymerase recognition sequences are identical in ColA, ColD, and ColE1 plasmids. In spite of the differences in the nucleotide sequences, RNA I and the preprimer RNA of ColA and ColD may form structures analogous to the respective structures of ColE1.

Bacteriocin Plasmids↗
PubMed1994

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein.

Replication of the gamma origin of Escherichia coli plasmid R6K requires pi protein, encoded by the R6K pir gene, and many host factors, including DnaA protein. Pi has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of pi is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using gamma origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type pi protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.

Bacterial Proteins↗
PubMed1980

Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication.

A DNA fragment containing the replication origin of the Eschericia coli K-12 chromosome was inserted in two correlations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA+ cells, whereas pBR322 replicates only in polA+ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function of the E. coli origin. In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori+ and Ori- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure. To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.

Base Sequence↗
PubMed2005

CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis.

Cyclin-dependent kinases (CDKs) restrict DNA replication origin firing to once per cell cycle by preventing the assembly of prereplicative complexes (pre-RCs; licensing) outside of G1 phase. Paradoxically, under certain circumstances, CDKs such as cyclin E-cdk2 are also required to promote licensing. Here, we show that CDK phosphorylation of the essential licensing factor Cdc6 stabilizes it by preventing its association with the anaphase promoting complex/cyclosome (APC/C). APC/C-dependent Cdc6 proteolysis prevents pre-RC assembly in quiescent cells and, when cells reenter the cell cycle from quiescence, CDK-dependent Cdc6 stabilization allows Cdc6 to accumulate before the licensing inhibitors geminin and cyclin A which are also APC/C substrates. This novel mechanism for regulating protein stability establishes a window of time prior to S phase when pre-RCs can assemble which we propose represents a critical function of cyclin E.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1983

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome.

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.

Base Sequence↗
PubMed1979

Cloning and expression of the Escherichia coli replication origin in a single-stranded DNA phage.

The Escherichia coli DNA replication origin (oriC) and the adjacent asparagine synthetase gene (asnA) have been inserted into the duplex replicative form DNA of the single-stranded phage vector M13Goril. By in vitro recombination, the entire oriC asnA-containing plasmid pJS5 was inserted into M13Gori1 in both possible orientations. Both phage types transduce the asnA gene and confer upon the M13 vector the ability to replicate as a plasmid in the E. coli mutant rep3. In rep+ hosts, these phages undergo single-stranded DNA synthesis and viral morphogenesis.

Cloning, Molecular↗
PubMed2003

Localization of proteins bound to a replication origin of human DNA along the cell cycle.

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site.

Base Sequence↗
PubMed2003

Genome-wide distribution of DNA replication origins at A+T-rich islands in Schizosaccharomyces pombe.

Genome-wide analysis of replication dynamics requires the previous identification of DNA replication origins (ORIs). However, variability among the ORIs makes it difficult to predict their distribution across the genome on the basis of their sequence. We report here that ORIs in Schizosaccharomyces pombe coincide with discrete chromosomal A+T-rich islands of up to 1 kb long that are characterized by a distinctive A+T content that clearly differentiates them from the rest of the genome. Genome-wide analysis has enabled us to identify 384 of these regions, which predicts the position of most ORIs in the genome, as shown by functional replication analyses. A+T-rich islands occur at the mating locus, centromeres and subtelomeric regions at a density that is approximately fourfold higher than elsewhere in the genome, which suggests a link between the origin recognition complex (ORC) and transcriptional silencing in these regions. The absence of consensus elements in A+T-rich islands implies that different sequences can target the ORC to different ORIs.

AT Rich Sequence↗
PubMed1987

Mapping of mycoplasma virus DNA replication origins and termini.

A pulse-labeling protocol has been used to study DNA replication and map replication origins and termini in mycoplasma viruses L2 and L2ins1. The L2 genome is circular, double-stranded DNA of 11.63 kilobase pairs (kb), and the 14.89-kb L2ins1 genome is L2 DNA containing a 3.26-kb insertion. The data show that DNA replication is bidirectional from two origins in L2 and three origins in L2ins1. The extra origin in L2ins1 arises from the fact that one of the L2 origins is in one of the sequences that have been shown to be duplicated and transposed in the generation of L2ins1 from L2.

Acholeplasma laidlawii↗
PubMed2001

Plasmids with a mammalian replication origin and a matrix attachment region initiate the event similar to gene amplification.

Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins.

Colorectal Neoplasms↗
PubMed2001

A broad replication origin of Drosophila melanogaster, oriDalpha, consists of AT-rich multiple discrete initiation sites.

This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU. Denatured DNA was size-fractionated in alkaline sucrose gradients. BrdU-labeled nascent DNA was immuno-affinity purified using anti-BrdU antibodies. DNA was quantified with a competitive PCR method before and after immuno-purification. The results indicated that oriDalpha, whose size was presumed to be about 10 kb from two-dimensional gel electrophoretic analysis, contained four major initiation sites in its central 2.8 kb region, and six to approximately eight sites in 8.4 kb. All initiation sites corresponded with AT-rich sequences. Detailed analysis of one major initiation site indicated that its range was restricted to 700 bp.

Animals↗
PubMed1988

A yeast replication origin consists of multiple copies of a small conserved sequence.

Autonomously replicating sequences (ARSs) of the yeast S. cerevisiae function as replication origins on plasmids and probably also on chromosomes. ARS function requires a copy of the ARS core consensus (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and additional sequences 3' to the T-rich strand of the consensus. Our analysis of an ARS from chromosome III, the C2G1 ARS, suggests that ARS function depends on the presence of an exact match to the core consensus and the presence of additional near matches in the 3' flanking region. We have demonstrated that ARS function can be mediated by multiple matches to the core consensus by constructing synthetic ARS elements from oligonucleotides containing copies of the consensus sequence. We find that two copies of the core consensus are sufficient for ARS activity and that an artificial ARS as efficient as a natural chromosomal ARS can be constructed from multiple core consensus elements in a specific orientation.

Base Sequence↗
PubMed1994

Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin.

We have determined a 180 kb contiguous sequence in the replication origin region of the Bacillus subtilis chromosome. Open reading frames (ORF) in this region were unambiguously identified from the determined sequence, using criteria characteristic for the B. subtilis gene structure, i.e., starting with an ATG, GTG or TTG codon preceded by sequences complementary to the 3' end of the 16S rRNA. Four rRNA gene sets, 7 individual tRNA genes and 1 scRNA gene were identified, occupying 20 kb in total. In the remaining 160 kb region, 158 ORFs were identified, suggesting that 1 ORF is coded on average by 1 kb of DNA of the B. subtilis genome. Among the 158 ORFs, the functions of 48 ORFs were assigned and those of 11 ORFs are suggested through significant similarities to known proteins present in data banks. However, the functions of more than half of the ORFs (63%) remain to be determined.

Amino Acid Sequence↗
PubMed1983

DNA replication origin of polyoma virus: early proximal boundary.

We constructed a series of deleted polyoma genomes by Bal 31 nuclease digestion from the unique Bg/I site at nucleotide 86 on the "early" side of the origin of DNA replication. The ability of the cloned deleted genomes to replicate was tested after transfection into mouse 3T6 fibroblasts or into the polyomatransformed C127 (COP5) mouse cell line (Tyndall et al., Nucleic Acids Res. 9:6231-6251, 1981). Deletions up to nucleotide 64-had no effect on the amount of replicated DNA accumulated, but larger deletions, extending up to nucleotide 42, decreased this amount 7- to 10-fold. By nucleotide 38, the quantity of detected DNA was down 100-fold, and by nucleotide 20, no replication could be detected. The minimum origin segment does not contain any known high-affinity, large tumor antigen binding site.

Animals↗