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At least 775 records · Page 43Linked to original sources
PubMed1977

Origin of replication, oriC, of the Escherichia coli chromosome: mapping of genes relative to R.EcoRI cleavage sites in the oriC region.

A precise genetic-physical map of the tna-ilv region at 82 min on the genetic map of E. coli is obtained through deletion mapping and analysis by restriction endonuclease EcoRI of plasmids, derived from an F' carrying the genes between aroE and ilv. A locus, designated het, which in its diploid state results in slow growth and heterogeneity of cell size due to distorted cell division, maps between bglB and asn, 30-45 kb counterclockwise of ilv. The pattern of R.EcoRI cleavage sites in the het region is identical with the pattern obtained by Marsh and Worcel (1977) who analyzed DNA labeled preferentially in the region of the DNA replication origin (oriC). We suggest that oriC is identical with the het site and that it can be allocated to a position 32 kb counterclockwise of the ilv operon.

Chromosome Mapping↗
PubMed1985

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: complexes with lambda O protein and with lambda O, lambda P, and Escherichia coli DnaB proteins.

The O protein of bacteriophage lambda is required for initiation of DNA replication at the lambda replicative origin designated ori lambda. The binding sites for O protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage lambda O protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage lambda O and P proteins and host DnaB protein interact at ori lambda to generate a larger structure than that formed by O protein alone; P and DnaB proteins fail to form any observable complex when O protein is excluded from the reaction mixture. We conclude that the specialized nucleoprotein structure formed by phage lambda O protein and ori lambda provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure. Specialized nucleoprotein structures may be a general means to confer exceptional accuracy on DNA transactions requiring extraordinary precision.

Bacterial Proteins↗
PubMed1982

Binding of the origin of replication of Escherichia coli to the outer membrane.

The replication origin of the Escherichia coli chromosome binds with high affinity to outer membrane preparations. This binding requires a 460 bp stretch of origin DNA between positions -40 and 420 of the oriC map. Specific binding can be detected by the use of a membrane filter retention assay in the presence of excess calf thymus DNA. This binding is enhanced by divalent cations and takes place specifically at a few (0.7-3.0) membrane sites per cell. The apparent affinity of origin DNA for membranes is enhanced by two peptides, (55 kilodaltons (kd) and 75 kd), which remain attached to the DNA through treatment with 5.5 M cesium chloride.

Bacterial Proteins↗
PubMed1996

Properties of a primer RNA-DNA hybrid at the mouse mitochondrial DNA leading-strand origin of replication.

Primers for vertebrate mitochondrial leading-strand DNA replication are products of transcription synthesized by mitochondrial RNA polymerase. The precursor primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed during transcription through the replication origin (Xu, B., and Clayton, D. A. (1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of this primer RNA intermediate, we have used two methods to reconstitute model R-loops containing the mouse mitochondrial DNA origin sequence. First, we demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop can be assembled by annealing presynthesized RNA and supercoiled DNA template in the presence of formamide. R-loop formation by either method is dependent on specific template sequences. The reconstituted R-loop is exceptionally stable and exhibits an unexpected structure. Structural studies indicate that the RNA strand is organized within the RNA-DNA base-paired region, suggesting that the heteroduplex interaction occurs through a specific conformation. We propose that the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery.

Animals↗
PubMed1991

Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication.

The DNA from a wide variety of human tumors has sustained mutations within the conserved p53 coding regions. We have purified wild-type and tumor-derived mutant p53 proteins expressed from baculovirus vectors and examined their interactions with SV40 DNA. Using DNAase I footprinting assays, we observed that both human and murine wild-type p53 proteins bind specifically to sequences adjacent to the late border of the viral replication origin. By contrast, mutant p53 proteins failed to bind specifically to these sequences. SV40 T antigen prevented wild-type p53 from interacting with this region. These data show that normal but not oncogenic forms of p53 are capable of sequence-specific interactions with viral DNA. Furthermore, they provide insights into the mechanisms by which viral proteins might regulate the control of viral growth and cell division.

Animals↗
PubMed1978

Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn.

Specialized transducing phages lambda asn harboring chromosomal DNA and genetic markers on either side of the asn gene were isolated. Phages carrying chromosomal DNA counterclockwise of the asn gene can upon infection establish themselves as self-replicating plasmids in asn, recA hosts lysogenic for lambda. It is concluded that this bypassing of normal lambda immunity is due to the presence of the chromosomal replication origin, oriC, in this class of phages. Genetic analysis and the determination of restriction endonuclease cleavage patterns of the different lambda asn lead to the allocation of oriC within 1.5 megadaltons of the asn gene towards the uncA, uncB genes at 82 min on the genetic map of E. coli. The clockwise order of genes on the chromosomes is found to be: bglB, (pst, glmS), (uncA, uncB), oriC, asn, trkD, rbs, rrnC, ilv.

Chromosome Mapping↗
PubMed1986

kDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication.

The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction.

Animals↗
PubMed1986

RNA terminating within the E. coli origin of replication: stringent regulation and control by DnaA protein.

RNA entering the E. coli replication origin, oriC, in the counterclockwise direction terminates at several sites throughout the origin sequence. The significant finding was that nine clusters of these termination sites are found at the nine clusters of RNA to DNA transitions in oriC. The majority of these transcripts terminates with cytosine. Termination sites are associated with 9 of the 11 GATC sites and all DnaA protein-binding sites. Chloramphenicol-treated cells contain an increased amount of this RNA species, while cells starved for isoleucine have greatly reduced levels, indicating that synthesis of these transcripts is stringently regulated. Both decreased and increased intracellular levels of DnaA protein decrease the fraction of transcription that enters oriC.

Bacterial Proteins↗
PubMed1989

Purification and characterization of a low-molecular-weight membrane protein with affinity for the Escherichia coli origin of replication.

A purification procedure was devised for a low-molecular-mass (about 10-kilodalton) membrane protein from Escherichia coli that was shown to bind specifically to the chromosomal replication origin region (oriC). Nitrocellulose membrane retention assays showed the binding site to be adjacent to the right boundary of the oriC minimal sequence. We determined the amino acid sequence of the N-terminal and C-terminal regions as well as the global amino acid composition of this membrane protein. Specific antibodies against the protein were produced and used to confirm the cell membrane location of the protein. These results demonstrate that this is a new membrane protein, different from the previously described B' protein, with specific binding activity for the oriC region. We propose that this protein be called membrane oriC-binding protein 2 (MOB2 protein).

Amino Acid Sequence↗
PubMed1981

Purification of bacteriophage lambda O protein that specifically binds to the origin of replication.

By means of a nitrocellulose filter binding assay, DNA binding activities among proteins fractionated from extracts of Escherichia coli carrying lambda dv have been surveyed. An activity was found that binds specifically to a fragment of 164 base pairs that specifies the lambda replication origin (lambda ori). This activity was not detected in an extract of cells not carrying the lambda dv plasmid. The activity was detected in extracts of cells carrying a hybrid plasmid in which the entire lambda O gene had been cloned and placed under the control of the lac promoter. Deletion of a 60 base pair segment in the 'amino-terminal region' of the O gene abolished this activity, indicating that the lambda ori binding protein is coded for by the lambda O gene. The ori-specific binding protein was purified by five fractionation steps. The most purified preparation consists of a major polypepide that migrates with a molecular weight of 32,000 in SDS-polyacrylamide gel electrophoresis. Binding of O protein to ori occurs in the absence of other protein aceous components.

Bacteriophage lambda↗
PubMed1989

In vivo studies of DnaA binding to the origin of replication of Escherichia coli.

We have shown that DnaA, a protein required for initiation of DNA replication in Escherichia coli, binds to three of four DnaA binding sequences in the replicative origin oriC (boxes R1, R2 and R4). Protein-oriC DNA interactions in minichromosomes carried by wild-type and dnaA mutant strains were demonstrated by in vivo footprinting using dimethylsulfate treatment of intact cells. The same characteristic enhancement/protection pattern was seen in wild-type minichromosomes or mutants defective in oriC function but carrying the four DnaA boxes. Minichromosomes in dnaA (Ts) mutants showed no protein binding at non-permissive temperatures and reduced binding even at permissive temperatures. In vivo footprints of the wild-type strain were identical to those obtained in vitro using purified DnaA proteins and oriC DNA. Transcription into oriC affected the binding of DnaA protein to the DnaA boxes. These findings suggest that the protein causing the in vivo footprints at oriC is DnaA.

Bacterial Proteins↗
PubMed1999

Activation of dormant origins of DNA replication in budding yeast.

Eukaryotic genomes often contain more potential replication origins than are actually used during S phase. The molecular mechanisms that prevent some origins from firing are unknown. Here we show that dormant replication origins on the left arm of budding yeast chromosome III become activated when both passive replication through them is prevented and the Mec1/Rad53 checkpoint that blocks late-origin firing is inactivated. Under these conditions, dormant origins fire very late relative to other active origins. These experiments show that some dormant replication origins are competent to fire during S phase and that passage of a replication fork through such origins can inactivate them.

Cell Cycle↗
PubMed2000

Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication.

An origin of replication (ori) was obtained from a naturally occurring beta-lactamase-producing plasmid isolated from Neisseria gonorrhoeae and used to construct shuttle vectors capable of replicating in N. gonorrhoeae, Haemophilus ducreyi, Haemophilus influenzae and Escherichia coli. Using the gonococcal proAB genes, we complemented proline-requiring N. gonorrhoeae F62 and E. coli HB101 in trans. The first demonstration of the expression of the green fluorescent protein (GFP) in either N. gonorrhoeae or H. ducreyi was shown using this vector, indicating that GFP may be a useful tool in the analysis of these organisms. This is the first report of a gonococcal vector based on a broad host range, genetically defined ori, and should facilitate the molecular analysis of gonococcal and Haemophilus genes.

Bacteria↗
PubMed2002

Mapping of early firing origins on a replication profile of budding yeast.

BACKGROUND: Understanding of the firing time determination of replication origins in the entire genome will require a genome-wide survey of replication origins and their mapping on chromosomes. A microarray technology was applied to obtain a genome-wide profile of DNA replication and to classify early firing origins. RESULTS: A total of 260 potential replication origins (PROs) were identified in the entire budding yeast genome: 247 as defined peaks on the replication profile and 13 as regions located in the chromosomal termini. Based on the firing time, the 247 PROs were classified into 143 early PROs and 104 late PROs, that were not randomly distributed on chromosomes but formed separated clusters. Most of the early PROs were found to fire in the presence of hydroxyurea, indicating that they were free from the control of the intra-S-checkpoint mediated by Mec1 and Rad53. CONCLUSIONS: The monitoring method of DNA replication and the analysis method of microarray data used in this study proved powerful for obtaining a genome-wide view of the initiation and progression of DNA replication.

Base Sequence↗
PubMed1992

Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.

We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.

Amino Acid Sequence↗
PubMed1997

Novel alleles of the Escherichia coli dnaA gene.

The Escherichia coli dnaA gene is required for replication of the bacterial chromosome. To identify residues critical for its replication activity, a method to select novel mutations was developed that relied on lytic growth of lambda from an inserted pSC101 replication origin. Replication from the lambda origin was inhibited by lysogen-encoded cI repressor. Replication from the pSC101 origin that resulted in lytic growth was dependent on active DnaA protein encoded by a plasmid in a host strain lacking the chromosomal dnaA gene. With this approach, a large collection of missense, nonsense, and a few internal deletion mutations were obtained. Nucleotide sequence analysis of the missense mutations indicated that 28 of 50 were unique. Of these, one was identical to the dnaA205 allele whereas the remainder are novel. These missense mutations were clustered into three regions, suggesting three functional domains of DnaA protein required for its replication activity. Many of the missense mutations mapping to the C-terminal 61 residues were inactive for replication from the pSC101 origin. These are defective in DNA binding. Mutations that mapped elsewhere were temperature-sensitive.

Alleles↗
PubMed2006

The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis.

Soj (ParA) and Spo0J (ParB) of Bacillus subtilis belong to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. Unlike most Par systems, for which intact copies of both parA and parB are required for the Par system to function, inactivating soj does not cause a detectable chromosome partitioning phenotype whereas inactivating spo0J leads to a 100-fold increase in the production of anucleate cells. This suggested either that Soj does not function like other ParA homologues, or that a cellular factor might compensate for the absence of soj. We found that inactivating smc, the gene encoding the structural maintenance of chromosomes (SMC) protein, unmasked a role for Soj in chromosome partitioning. A soj null mutation dramatically enhanced production of anucleate cells in an smc null mutant. To look for effects of a soj null on other phenotypes perturbed in a spo0J null mutant, we analysed replication initiation and origin positioning in (soj-spo0J)+, Deltasoj, Deltaspo0J and Delta(soj-spo0J) cells. All of the mutations caused increased initiation of replication and, to varying extents, affected origin positioning. Using a new assay to measure separation of the chromosomal origins, we found that inactivating soj, spo0J or both led to a significant defect in separating replicated sister origins, such that the origins remain too close to be spatially resolved. Separation of a region outside the origin was not affected. These results indicate that there are probably factors helping to pair sister origin regions for part of the replication cycle, and that Soj and Spo0J may antagonize this pairing to contribute to timely separation of replicated origins. The effects of Deltasoj, Deltaspo0J and Delta(soj-spo0J) mutations on origin positioning, chromosome partitioning and replication initiation may be a secondary consequence of a defect in separating replicated origins.

Bacillus subtilis↗