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PubMed1986

Dissection of promoter sequences involved in transcriptional activation of the Escherichia coli replication origin.

The replication frequency of oriC plasmids in vivo is positively affected by specific transcripts running into oriC. These transcripts that activate oriC are initiated at a promoter of a gene coding for a 16kD protein. Genetic evidence is presented for binding of the initiation factor dnaA to a specific sequence (dnaA box) upstream of this promoter. Binding of the dnaA protein to this dnaA box regulates transcription initiation negatively. It was also demonstrated that binding of dnaA protein to the 16kD promoter region is essential to accomplish the actual activation event within the origin. Replication and incompatibility experiments suggest that dnaA protein is present within the activating transcription complex. The function of dnaA in this replication control mechanism is discussed.

Bacterial Proteins↗

PubMed2001

A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1.

As in other yeasts, ARS-containing plasmids can be maintained extrachromosomally in Kluyveromyces lactis. Although some fragments of K. lactis DNA have ARS activity in both K. lactis and Saccharomyces cerevisiae, it appears that the sequences required for ARS activity in the two yeasts are different. As an approach to a better understanding of ARS structure and function in K. lactis, we analyzed the replication of the circular plasmid pKD1. We identified a 159-bp sequence able to promote autonomous replication of pKD1 in both yeasts; this fragments contains both a sequence related to the S. cerevisiae ARS consensus sequence and a region of 53% identity to the 40-bp sequence essential for K. lactis KARS101 function. By the analysis of in vivo replication intermediates we provide the first direct evidence that DNA replication initiates at or near the K. lactis ARS element. Replication terminates at the cisacting stability locus of pKD1, which functions as a replication fork barrier (RFB) and is necessary for proper plasmid segregation. RFB activity requires the pKDI gene products that are important for plasmid segregation, suggesting a link between DNA replication termination and plasmid segregation in a eukaryotic organism.

Base Sequence↗

PubMed2001

Making sense of eukaryotic DNA replication origins.

DNA replication is the process by which cells make one complete copy of their genetic information before cell division. In bacteria, readily identifiable DNA sequences constitute the start sites or origins of DNA replication. In eukaryotes, replication origins have been difficult to identify. In some systems, any DNA sequence can promote replication, but other systems require specific DNA sequences. Despite these disparities, the proteins that regulate replication are highly conserved from yeast to humans. The resolution may lie in a current model for once-per-cell-cycle regulation of eukaryotic replication that does not require defined origin sequences. This model implies that the specification of precise origins is a response to selective pressures that transcend those of once-per-cell-cycle replication, such as the coordination of replication with other chromosomal functions. Viewed in this context, the locations of origins may be an integral part of the functional organization of eukaryotic chromosomes.

Animals↗

PubMed2003

A multifunctional plasmid-encoded replication initiation protein both recruits and positions an active helicase at the replication origin.

The DnaA replication initiation protein has been shown to be essential for DNA strand opening at the AT-rich region of the replication origin of the Escherichia coli chromosome as well as serving to recruit and position the DnaB replicative helicase at this open region. Homologues of the dnaA gene of E. coli have been found in most bacterial species, and the DnaA protein has been shown to be required for the initiation of replication of both chromosomal and plasmid DNA. For several plasmid elements it has been found that a plasmid-encoded initiation protein is required along with the DnaA protein to bring about opening of the AT-rich region at the replication origin. The broad host range plasmid RK2 encodes two forms of its replication initiation protein (TrfA-33 and TrfA-44) that differ by an additional 98 aa at the N terminus of the larger (TrfA-44) form. Both forms initiate replication of RK2 in E. coli in vitro by a DnaA-dependent mechanism. However, as shown in this study, TrfA-44 specifically interacts with the DnaB replicative helicase of Pseudomonas putida and Pseudomonas aeruginosa and initiates the formation of a prepriming open complex in the absence of DnaA protein. Thus, the TrfA-44 initiation protein has the multifunctional properties of recruiting and positioning an active form of the DnaB helicase at the RK2 replication origin by a DnaA-independent process. This unique property for a replication initiation protein undoubtedly plays an important role in extending the host range of the RK2 antibiotic resistance plasmid.

Bacterial Proteins↗

PubMed2000

Polymorphism and divergence in the beta-globin replication origin initiation region.

DNA sequence polymorphism and divergence was examined in the vicinity of the human beta-globin gene cluster origin of replication initiation region (IR), a 1.3-kb genomic region located immediately 5' of the adult-expressed beta-globin gene. DNA sequence variation in the replication origin IR and 5 kb of flanking DNA was surveyed in samples drawn from two populations, one African (from the Gambia, West Africa) and the other European (from Oxford, England). In these samples, levels of nucleotide and length polymorphism in the IR were found to be more than two times as high as adjacent non-IR-associated regions (estimates of per-nucleotide heterozygosity were 0.30% and 0.12%, respectively). Most polymorphic positions identified in the origin IR fall within or just adjacent to a 52-bp alternating purine-pyrimidine ((RY)n) sequence repeat. Within- and between-populations divergence is highest in this portion of the IR, and interspecific divergence in the same region, determined by comparison with an orthologous sequence from the chimpanzee, is also pronounced. Higher levels of diversity in this subregion are not, however, primarily attributable to slippage-mediated repeat unit changes, as nucleotide substitution contributes disproportionately to allelic heterogeneity. An estimate of helical stability in the sequenced region suggests that the hypervariable (RY)n constitutes the major DNA unwinding element (DUE) of the replication origin IR, the location at which the DNA duplex first unwinds and new strand synthesis begins. These findings suggest that the beta-globin IR experiences a higher underlying rate of neutral mutation than do adjacent genomic regions and that enzyme fidelity associated with the initiation of DNA replication at this origin may be compromised. The significance of these findings for our understanding of eukaryotic replication origin biology is discussed.

Animals↗

PubMed2001

Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins.

BACKGROUND: Eukaryotic DNA replication is initiated from distinct regions on the chromosome. However, the mechanism for recognition of replication origins is not known for most eukaryotes. In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs). Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found. In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC. RESULTS: The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome. SpORC-IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity. The nucleotide sequences required for the interaction with SpORC-IPs correspond closely to those necessary for in vivo ARS activity. CONCLUSION: Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin. The presence of multiple SpORC-binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins.

Adenine↗

PubMed2004

The activities of eukaryotic replication origins in chromatin.

DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit replication proteins with the potential to form context-dependent protein-protein interactions.

Acetylation↗

PubMed1981

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. II. Identification of the essential regions for inhibitory functions shown by the DNA segment containing the replication origin.

A BamHI restriction endonuclease fragment B7, which contains the replication origin of the Bacillus subtilis chromosome, showed inhibitory effects on cell growth and plasmid replication in Escherichia coli and Bacillus subtilis, when B7 was inserted into a composite plasmid pMS102' and introduced into these cells. In order to localize these properties in more limited regions within the B7 fragment, we developed a new and widely applicable method for deletion of DNA segments of various lengths from one or other end of a given region of the plasmid molecule. Using a set of deletions in the B7 fragments of pMS102'-B7, we determined the loci responsible for the inhibitory effects of B7 as described below. (1) Stickiness appearing in E. coli cells was caused by a segment residing in a region of approximately 2.2 kilobase pairs (kbp) overlapping the E19 and E22 fragments. (2) instability of the plasmid in E. coli was due to a segment localized in the 440 bp region of the E19-side terminal portion of the 2.2 kbp region. (3) The same 440 bp were also responsible for inhibition of the replication of the plasmid in B. subtilis. Hybridization of the cloned DNA fragments containing the 2.2 kbp region with the whole B. subtilis chromosome revealed that several regions of the chromosome are homologous to this characteristic sequence.

Bacillus subtilis↗

PubMed1993

Plasmids from two morphologically distinct cyanobacterial strains share a novel replication origin.

A 2.9-kbp replication origin from a plasmid endogenous to the filamentous cyanobacterium Fremyella diplosiphon UTEX 481 was genetically characterized and sequenced. Deletion analysis of the 2.9-kbp DNA fragment delimited the minimum region necessary for replication in F. diplosiphon Fd33 to approximately 2.5 kbp. DNA sequence analysis revealed that the F. diplosiphon plasmid replication origin is structurally very similar to and shares significant identity with the 1.75-kbp replication origin reported for plasmid pDU1, isolated from the morphologically distinct cyanobacterium Nostoc sp. strain PCC 7524. Each cyanobacterial plasmid replication origin includes a large open reading frame that predicts a conserved protein of unknown function; the predicted proteins of the replication origins are of similar sizes and 30% identical in amino acid sequence. Each cyanobacterial plasmid replication origin also possesses a region of dyad symmetry approximately 300 bp upstream of the conserved open reading frame.

Amino Acid Sequence↗

PubMed1991

Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins.

Four Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from purified P. hybrida mitochondria, specific initiation of DNA synthesis could only be observed starting within two fragments, oriA and oriB. When DNA synthesis incubations were performed with DNA templates consisting of both the A and B origins in the same plasmid in complementary strands, DNA synthesis first initiates in the A-origin, proceeds in the direction of the B-origin after which replication is also initiated in the B-origin. Based on these observations, a replication model for the P. hybrida mitochondrial genome is presented.

Animals↗

PubMed1989

Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.

Adenine↗

PubMed1993

Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin.

The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, E1 and E2, while all other proteins are derived from the host. We described previously the isolation of the E1 protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The E1 protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maximal efficiency. The E1 protein also catalyzed a BPV origin-dependent DNA unwinding reaction. In this report, we show that at low levels of E1 protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by E1, but it had no effect on the nonspecific E1-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the E1 protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing E1 binding to the BPV origin via DNA-protein and protein-protein interactions.

Animals↗

PubMed1997

Functional equivalency and diversity of cis-acting elements among yeast replication origins.

The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.

DNA Helicases↗

PubMed2004

Prediction of Saccharomyces cerevisiae replication origins.

BACKGROUND: Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible. RESULTS: We developed an algorithm, Oriscan, to predict yeast origins using similarity to 26 characterized origins. Oriscan used 268 bp of sequence, including the T-rich ACS and a 3' A-rich region. The predictions identified the exact location of the ACS. A total of 84 of the top 100 Oriscan predictions, and 56% of the top 350, matched known ARSs or replication protein binding sites. The true accuracy was even higher because we tested 25 discrepancies, and 15 were in fact ARSs. Thus, 94% of the top 100 predictions and an estimated 70% of the top 350 were correct. We compared the predictions to corresponding sequences in related Saccharomyces species and found that the ACSs of experimentally supported predictions show significant conservation. CONCLUSIONS: The high accuracy of the predictions indicates that we have defined near-sufficient conditions for ARS activity, the A-rich region is a recognizable feature of ARS elements with a probable role in replication initiation, and nucleotide sequence is a reliable predictor of yeast origins. Oriscan detected most origins in the genome, demonstrating previously unrecognized generality in yeast replication origins and significant discriminatory power in the algorithm.

Algorithms↗

PubMed1999

Genetic methods for characterizing the cis-acting components of yeast DNA replication origins.

Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species. Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function. Although alternative methods are available for genetic characterization of replication origins in other organisms, the simplicity of the autonomous replication assay in yeasts has permitted development of the deepest understanding to date of eukaryotic replication origin structure. This information has come primarily from studies with Saccharomyces cerevisiae. However, there are many other yeast species, each with its own variety of replication origins. Use of the methods summarized here to characterize origins in other yeast species is likely to provide additional insights into eukaryotic replication origin structure.

Cell Division↗

PubMed1990

Identification of critical elements within the JC virus DNA replication origin.

The T antigen of JC virus (JCV) does not interact productively with the simian virus 40 (SV40) origin of replication. In contrast, the SV40 T antigen does drive replication from the JCV origin as well as from its own. The basis for this restricted interaction was investigated by analyzing the structure of the JCV replication origin. The replication activities of JCV-SV40 hybrid origin plasmids were tested in cells constitutively producing either the JCV or SV40 T antigen. Results indicated that a region of the JCV origin critical for interaction with the JCV T antigen was positioned to the late side of the central palindrome of the putative core origin. A mutational analysis of this region indicated that the sequence of the A + T-rich tract was primarily responsible for determining the efficiency with which JCV can initiate replication from its origin. The tandemly repeated pentameric sequence AGGGA located proximal to the A + T-rich tract in the JCV enhancer element was found to stimulate JCV, but not SV40, T antigen-mediated replication. The effect on replication of other elements within the JCV enhancer was also dependent on the T antigen employed for initiation. A plasmid containing the replication origin of prototype BK virus was unable to replicate in cells containing JCV T antigen, again indicating the inflexibility of the JCV T antigen in interacting with heterologous origins.

Animals↗

PubMed1995

S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state.

BACKGROUND: DNA replication and mitosis are triggered by activation of kinase complexes, each made up of a cyclin and a cyclin-dependent kinase (Cdk). It had seemed possible that the association of Cdks with different classes of cyclins specifies whether S phase (replication) or M phase (mitosis) will occur. The recent finding that individual B-type cyclins (encoded by the genes CLB1-CLB6) can have functions in both processes in the budding yeast Saccharomyces cerevisiae casts doubt on this notion. RESULTS: S. cerevisiae strains lacking C1b1-C1b4 undergo DNA replication once but fail to enter mitosis. We have isolated mutations in two genes, SIM1 and SIM2 (SIM2 is identical to SEC72), which allow such cells to undergo an extra round of DNA replication without mitosis. The Clb5 kinase, which promotes S phase, remains active during the G2-phase arrest of cells of the parental strain, but its activity declines rapidly in sim mutants. Increased expression of the CLB5 gene prevents re-replication. Thus, a cyclin B-kinase that promotes DNA replication in G1-phase cells can prevent re-replication in G2-phase cells. Inactivation of C1b kinases by expression of the specific C1b-Cdk1 inhibitor p40SIC1 is sufficient to induce a prereplicative state at origins of replication in cells blocked in G2/M phase by nocodazole. Re-activation of C1b-Cdk1 kinases induces a second round of DNA replication. CONCLUSIONS: We propose that S-phase-promoting cyclin B--Cdk complexes prevent re-replication during S, G2 and M phases by inhibiting the transition of replication origins to a pre-replicative state. This model can explain both why origins 'fire' only once per S phase and why S phase is dependent on completion of the preceding M phase.

Cell Cycle↗

PubMed1997

Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins.

5-Methylcytosine↗