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PubMed1995

Clusters of modular regulatory elements at DNA replication origins.

To study the specificity of eukaryotic origins of replication (ori), we have isolated a fraction of mouse DNA enriched in replication initiation sequences (RIS), and cloned and characterised some of these RIS. The sequences of three RIS were analysed for the presence of sequence elements common to other known eukaryotic ori. It was found that the three RIS were A+T rich and contained a number of sequence elements that may function in the initiation of DNA replication. The data support the idea that mammalian ori are organised from modular sequence elements.

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↗
PubMed1999

Phage phi29 terminal protein residues Asn80 and Tyr82 are recognition elements of the replication origins.

Initiation of phage phi29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the phi29 terminal protein (TP) and the phi29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various phi29 TP mutants were generated at conserved amino acid residues from positions 61 to 87. In vitro phi29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with phi29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP.

Amino Acid Sequence↗
PubMed1999

Association of fission yeast Orp1 and Mcm6 proteins with chromosomal replication origins.

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.

Cell Cycle↗
PubMed2000

Interactions of DnaA proteins from distantly related bacteria with the replication origin of the broad host range plasmid RK2.

Replication initiation of the broad host range plasmid RK2 requires binding of the host-encoded DnaA protein to specific sequences (DnaA boxes) at its replication origin (oriV). In contrast to a chromosomal replication origin, which functionally interacts only with the native DnaA protein of the organism, the ability of RK2 to replicate in a wide range of Gram-negative bacterial hosts requires the interaction of oriV with many different DnaA proteins. In this study we compared the interactions of oriV with five different DnaA proteins. DNase I footprint, gel mobility shift, and surface plasmon resonance analyses showed that the DnaA proteins from Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa bind to the DnaA boxes at oriV and are capable of inducing open complex formation, the first step in the replication initiation process. However, DnaA proteins from two Gram-positive bacteria, Bacillus subtilis and Streptomyces lividans, while capable of specifically interacting with the DnaA box sequences at oriV, do not bind stably and fail to induce open complex formation. These results suggest that the inability of the DnaA protein of a host bacterium to form a stable and functional complex with the DnaA boxes at oriV is a limiting step for plasmid host range.

Amino Acid Sequence↗
PubMed1999

Organization of DNA replication origins in the fission yeast genome.

Eukaryotic DNA replication initiates at multiple points along the chromosomes known as replication origins (ORIs). We have developed a strategy to identify ORIs directly from replication intermediates in the fission yeast Schizosaccharomyces pombe. Mapping of a selection of the novel ORIs onto the genome reveals their preferential localization at intergenic regions upstream from genes. These results are supported by the observation that a large proportion of regions overlapping gene promoters contain active ORIs. Mapping of the genomic ars1 replication origin at nucleotide resolution shows that replication initiates at a defined position immediately upstream from the hus5(+) promoter. Deletion analysis indicates that the regulatory elements required to initiate transcription and replication lie in close proximity, suggesting a possible relationship between both processes in vivo.

Base Sequence↗
PubMed1984

Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena.

The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.

Animals↗
PubMed1986

Permanent attachment of replication origins to the nuclear matrix in BHK-cells.

The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.

Animals↗
PubMed1998

Characterization of a plasmid replicative origin from an extreme thermophile.

The minimal replicative origin from a 16-kbp natural plasmid of Thermus sp. ATCC 27737 is described. The 1798-bp sequence encodes a single protein (RepA) whose expression is required for replication. This requirement is extended to 200 bp upstream the coding region, which contains promoter-like sequences transcriptionally active in E. coli. Despite the absence of typical iterons, we show that an N-terminal fragment of this protein binds to a DNA fragment internal to its own coding sequence. As this DNA fragment contains an unusually A + T rich sequence surrounded by C + G rich stretches, the possible location of the replicative origin within the region encoding RepA is proposed.

Amino Acid Sequence↗
PubMed1994

Eukaryotic replication origins: control in space and time.

Replication origins facilitate the choreography of genome duplication by acting as targets for regulatory mechanisms. Eukaryotic cells control the efficiency and the time of origin activity. In addition, origins that have been replicated are prevented from doing so twice in the same cell cycle. In this review we will examine the mechanisms that may be used to control these processes, and discuss the role of replication in regulating transcription.

Animals↗
PubMed1999

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Adenosine Triphosphate↗
PubMed1980

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence from about 100 base-pairs downstream to about 600 base pairs upstream the CloDF13 replication origin has been determined. A comparison of this sequence with the corresponding ColE1 origin sequence reveals that: The sequence at the origin of replication is conserved. There are large differences in the nucleotide sequence downstream the replication origin, whereas there is a large homology in the region of about 410 base-pairs upstream the replication origin. This conserved region might code for a largely homologous basic, arginine rich polypeptide of about 45 amino-acids, for both ColE1 and CloDF13. Although there are large differences in the primary structure of the region coding for the 100 nucleotide RNA, the secondary structure of this region seems to be conserved.

Base Composition↗
PubMed2000

Think global, act local--how to regulate S phase from individual replication origins.

All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.

Animals↗
PubMed1990

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.

Animals↗
PubMed1988

Deletion analysis of bacteriophage T4 tertiary origins. A promoter sequence is required for a rifampicin-resistant replication origin.

The DNA sequence requirements of two T4 tertiary replication origins have been characterized by a deletion analysis of origin-containing plasmids. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 base pairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.

Base Sequence↗
PubMed1995

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.

Animals↗
PubMed1994

A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae.

We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast. This was done by substituting the chromosomal copy with a series of ars1 deletion mutants. It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains. We conclude that replication origins are abundantly, present in the yeast genome and that the removal of a single replication origin is compensated for by replication forks emanating from neighbouring origins.

Chromosomes, Fungal↗
PubMed1990

[Cloning and study on the replication origin of the integrated F plasmid in Escherichia coli].

The replication origin of the integrated F' plasmid was cloned by means of marker rescue. No difference in incompatibility and acridine orange sensitivity was found between the mini-F plasmid constructed from such an origin and the autonomous F' plasmid. Subcloning and comparative restriction enzyme analysis were carried out with the replication origin from the integrated F' plasmid and that from the autonomous F plasmid. No structural difference was found between them. These results suggest that the difference for the dependence of recA gene between the F and F' plasmids dose not come from any difference in structure of their replication origin, but probably from their site of integration on the chromosome.

Cloning, Molecular↗