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At least 253 records · Page 14Linked to original sources
PubMed1997

A transcriptional insulator element, the su(Hw) binding site, protects a chromosomal DNA replication origin from position effects.

Eukaryotic chromosomes are organized into domains of activity for both transcription and DNA replication. Transcriptional "border," or "insulator," elements have been implicated in mediating the organization of transcriptional domains. However, the DNA sequence elements which might demarcate domains of DNA replication activity are unknown. su(Hw) protein binding sites [su(Hw)BSs] are potent transcriptional insulator elements which can block enhancer action, as well as positive and negative chromosomal position effects. Here we report that flanking su(Hw)BSs can also create a chromosomal domain permissible for activity of the chorion gene DNA replication origin. During Drosophila oogenesis the chorion (eggshell) gene loci are amplified approximately 80-fold through repeated initiation of DNA replication. The cis-acting amplification control element, on the third chromosome (ACE3), is required for high levels of amplification initiating at the nearby major origin of replication, Ori-beta. A transgenic chorion locus construct containing ACE3 and Ori-beta was able to amplify but was extremely sensitive to position effects: only 7 of 21 independent insertions amplified >10-fold. The inclusion of flanking su(Hw)BSs in the construct dramatically protected DNA replication from position effects: 31 of 31 insertions now amplified >10-fold, and this protection was reduced in a su(Hw) mutant background. Amplification was equal on both sides of the su(Hw)BS, demonstrating that replication fork passage is not significantly impeded by these sites. Inclusion of only a single su(Hw)BS in the construct did not detectably protect the chorion gene DNA replication origin from position effects.

Animals↗
PubMed2003

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA.

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli

Bacterial Proteins↗
PubMed1990

Pseudomonas chromosomal replication origins: a bacterial class distinct from Escherichia coli-type origins.

The bacterial origins of DNA replication have been isolated from Pseudomonas aeruginosa and Pseudomonas putida. These origins comprise a second class of bacterial origins distinct from enteric-type origins: both origins function in both Pseudomonas species, and neither functions in Escherichia coli; enteric origins do not function in either pseudomonad. Both cloned sequences hybridize to chromosomal fragments that show properties expected of replication origins. These origin plasmids are highly unstable, are present at low copy number, and show mutual incompatibility properties. DNA sequence analysis shows that both origins contain several 9-base-pair (bp) E. coli DnaA protein binding sites; four of these are conserved in position and orientation, two of which resemble the R1 and R4 sites of the E. coli origin. Conserved 13-bp direct repeats adjacent to the analogous R1 site are also found. No GATC sites are in the P. aeruginosa origin and only four are in the P. putida origin; no other 4-bp sequence is present in high abundance. Both origins are found between sequences similar to the E. coli and Bacillus subtilis dnaA, dnaN, rpmH, and rnpA genes, a gene organization identical to that for B. subtilis and unlike that of E. coli. A second autonomously replicating sequence was obtained from P. aeruginosa that has some properties of bacterial origins.

Base Sequence↗
PubMed1983

Organization of DNA sequences and replication origins at yeast telomeres.

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.

Base Sequence↗
PubMed2006

Mapping of DNA replication origins to noncoding genes of the X-inactivation center.

In mammals, few DNA replication origins have been identified. Although there appears to be an association between origins and epigenetic regulation, their underlying link to monoallelic gene expression remains unclear. Here, we identify novel origins of DNA replication (ORIs) within the X-inactivation center (Xic). We analyze 86 kb of the Xic using an unbiased approach and find an unexpectedly large number of functional ORIs. Although there has been a tight correlation between ORIs and CpG islands, we find that ORIs are not restricted to CpG islands and there is no dependence on transcriptional activity. Interestingly, these ORIs colocalize to important genetic elements or genes involved in X-chromosome inactivation. One prominent ORI maps to the imprinting center and to a domain within Tsix known to be required for X-chromosome counting and choice. Location and/or activity of ORIs appear to be modulated by removal of specific Xic elements. These data provide a foundation for testing potential relationships between DNA replication and epigenetic regulation in future studies.

Animals↗
PubMed2002

Increased recombination intermediates and homologous integration hot spots at DNA replication origins.

We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis. Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids. The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site. The S. pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates. These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.

DNA Replication↗
PubMed1999

A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin.

The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes. Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex. Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes. However, DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication.

Bacterial Proteins↗
PubMed1994

Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function.

ARS307 is highly active as a replication origin in its native location on chromosome III of Saccharomyces cerevisiae. Its ability to confer autonomous replication activity on plasmids requires the presence of an 11-bp autonomously replicating sequence (ARS) consensus sequence (ACS), which is also required for chromosomal origin function, as well as approximately 100 bp of sequence flanking the ACS called domain B. To further define the sequences required for ARS function, a linker substitution mutagenesis of domain B was carried out. The mutations defined two sequences, B1 and B2, that contribute to ARS activity. Therefore, like ARS1, domain B of ARS307 is composed of functional subdomains. Constructs carrying mutations in the B1 element were used to replace the chromosomal copy of ARS307. These mutations caused a reduction in chromosomal origin activity, demonstrating that the B1 element is required for efficient chromosomal origin function.

Base Sequence↗
PubMed1980

Site-specific deletion at the replication origin of the antibiotic resistance factor R1.

The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.

Anti-Bacterial Agents↗
PubMed2006

Replication-associated strand asymmetries in vertebrate genomes and implications for replicon size, DNA replication origin, and termination.

Strand compositional asymmetry has been observed in prokaryotes and used in predicting prokaryotic DNA replication origins and termini. However, it was not found in eukaryotic genomes by the same methods. We propose that transcription-associated strand asymmetries mask the replication-associated ones. By analyzing the nucleotide composition of intergenic sequences larger than 50 kb by cumulative skew diagrams (CSD), we found replication-associated strand asymmetry in vertebrate genomes. Furthermore, we found that the most common replicon sizes in vertebrates are 50-100 kb, and show evidence that the replication origin and termination regions of vertebrate genomes range from a discrete site to a broad zone.

Animals↗
PubMed1998

Cdc7 is required throughout the yeast S phase to activate replication origins.

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7(ts) allele were first arrested in G1 at the cdc7 block by incubation at 37 degrees C, and then were allowed to enter S phase by brief incubation at 23 degrees C. During the S phase, after return to 37 degrees C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.

Cell Cycle Proteins↗
PubMed2005

An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene.

Early in female mammalian embryogenesis, one of the two X chromosomes is inactivated to compensate the gene dosage between males and females. One of the features of X chromosome inactivation (XCI) is the late replication of the inactivated X chromosome. This study reports the identification, by competitive PCR of nascent DNA, of a replication origin in intron 2 of the human X-linked HPRT gene, that is functional only on the inactive X. Features frequently associated with replication origins, including a peak of enhanced DNA flexibility, a perfect match to the yeast ACS sequence, a 14/15 match to the Drosophila topoisomerase II consensus, and a 20/21 match to an initiation region consensus sequence, were identified close to the replication origin. The origin is located approximately 2 kb upstream of a matrix attachment region (MAR) and also contains two A:T-rich elements, thought to facilitate DNA unwinding.

Animals↗
PubMed1982

The nucleotide sequence of the replication origin beta of the plasmid R6K.

We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta. This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature. The nucleotide sequence of the region of replication origin beta has been determined. The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.

Base Composition↗
PubMed1996

Strain-dependent differences in the human cytomegalovirus replication origin.

The nucleotide sequence of the human cytomegalovirus replication origin of strain Towne (an AatII-SacI fragment corresponding to nt 90372-94637 of strain AD169) was determined and compared with AD169. Two differences were found in the nucleotide sequence level. One was the alteration of structural organization (a major difference): a 189-bp region of AD169 (nt 93337-93525) was directly repeated three times in Towne. The other was a change in the nucleotide residue level including substitution, insertion, or deletion (a minor difference). The divergent residues were predominantly localized within the nt 92591-92855 region of AD169. A replication assay revealed that replication ability remained after deletion of the 189-bp repeat but disappeared after either a 1.5-kb deletion from the AatII end or a 0.9-kb deletion from the SacI end. The 1.5- and 0.9-kb regions were relatively conserved. These results indicate that at least two regions essential for replication ability lie outside of both the relatively variable region and the 189-bp repeat and suggest that these essential regions support replication even with a spatial separation of either one (AD169) or three repeats (Towne) of the 189-bp region.

Base Sequence↗
PubMed1983

Effect of a stem-loop structure within the SV40 replication origin upon SV40 T antigen binding to origin region sequences.

Heteroduplexes were formed between SV40 replication origin-containing DNA fragments derived from wild-type genomic DNA and a viral deletion mutant (dl295) lacking 18 base pairs from a 27 bp inverted repeat that is imbedded within the minimal replication origin sequence. Among these structures were molecules bearing a 7 bp stem and a 3 nucleotide loop on the wild-type strand between nucleotides 5230 and 6. By nondenaturing gel electrophoresis, heteroduplex molecules bearing such a structure on the E-strand could be separated from those with the complementary structure on the L-strand, and either heteroduplex could be so separated from either the wild-type or dl295 homoduplex fragment. Each of these forms was singly 5' or 3' end-labeled and then evaluated as a target for specific binding to purified SV40 large T antigen isolated from wild-type virus-infected cells. The results of DNAase footprint protection analyses showed that the existence of both the E- and L-strand stem-loop structures inhibited T binding to site 2 which composes much of the minimal origin sequence. By contrast, T bound readily to this site in both homoduplexes. Furthermore, T protected both E- and L-strand sequences of its strongest binding site (site 1)--which abuts the early side of site 2--when the site 2 stem-loop was on the L-strand, and protected L-strand site 1 sequences normally when the stem-loop was on the E-strand. However, a marked alteration of binding to site 1 E-strand sequences was noted when the stem-loop was on the E-strand. Thus this alteration in replication origin secondary structure resulted in discrete local and vicinal effects on T binding. Furthermore, the results suggest that within site 1--a sequence employed as an early transcriptional regulatory locus--T antigen can bind specifically and tightly to one strand without exhibiting similar behavior on the other strand.

Antigens, Viral, Tumor↗
PubMed2005

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32.

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.

Actinobacteria↗
PubMed1991

Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

Chromosome Mapping↗
PubMed2005

Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding.

Triggering new rounds of chromosomal DNA replication during the bacterial cell cycle is exquisitely regulated, ensuring both proper timing and one round per cycle stringency. A critical first step is stable unwinding of oriC, the chromosomal replication origin, by multiprotein orisome complexes comprising the AAA+ initiator DnaA and modulator proteins that bend DNA. Recently identified oriC-DnaA interactions in Escherichia coli raise important questions regarding the molecular mechanisms that regulate origin unwinding in bacteria. We describe staged binding of E. coli origin recognition proteins and suggest an unwinding switch based on interactions between DnaA-ATP and specialized oriC sites that must be filled during orisome assembly. By focusing multiple regulatory pathways on only a few key oriC DNA-protein interactions, this model includes an efficient way to control unwinding followed by orisome inactivation during the cell cycle. Future studies will determine whether this regulatory scheme is correct and whether it is generally applicable to other bacterial types.

Chromosomes, Bacterial↗