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PubMed1991

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.

Amino Acid Sequence↗
PubMed1984

The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites.

Specific binding of dnaA protein to the E. coli origin of replication (oriC) is essential for initiation of chromosomal replication. Based on binding of plasmid DNA fragments, dnaA protein recognizes in addition to oriC a number of specific sites: within or near the replication origins of pSC101, pBR322, and ColE1; within the regulatory regions of the dnaA and "X-protein" genes; and in IRL-Tn5. All strong binding sites share a common 9 bp sequence, 5'-TTATCACACAA, repeated at four conserved positions within oriC. As shown by DNAase I footprinting, dnaA protein binding is highly cooperative, covering 250 bp at oriC and 100 bp at single 9 bp sequence sites. Consistent with filter-binding and nuclease-protection studies, complexes of 20 to 30 dnaA monomers are visualized at oriC and other sites by electron microscopy. The functions of dnaA complexes and 9 bp sequences at these sites are discussed.

Bacterial Proteins↗
PubMed1990

T-antigen is not bound to the replication origin of the simian virus 40 late transcription complex.

Simian virus 40 tumor antigen (T-antigen) plays a central role in determining which gene is transcribed from viral DNA late in infection. Results from several studies have led to a model in which the binding of T-antigen to the viral origin of replication results in repression of transcription from the stronger early gene promoter and stimulation of transcription from the late gene promoter. We have tested this model by determining directly the occupancy of the T-antigen binding site in the origin of replication of the late transcription complex. Thus, viral transcription complexes were digested with BglI, a restriction enzyme that cuts in the viral replication origin. The enzyme cleaved 78(+/- 12)% of the late transcription complexes. Control experiments demonstrated that cleavage is blocked when T-antigen is bound to the origin site, that exogenously added T-antigen can bind to the site in the transcription complex, and that T-antigen is not released during isolation of the complex. These results indicate that most of the late transcription complexes do not have T-antigen bound to the origin site, and are therefore inconsistent with models that require this site to be occupied by T-antigen to maintain proper regulation of gene transcription late in infection.

Animals↗
PubMed1987

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.

Alleles↗
PubMed1992

Interaction with the nuclear matrix of a chimeric construct containing a replication origin and a transcription unit.

We have studied the interaction of a chimeric construct containing an origin of replication (from bovine papilloma virus) and a hormonally regulated transcription unit (long terminal repeat from the mouse mammary tumor virus, driving the v-Ha-ras gene) with the nuclear scaffold and matrix from mouse fibroblasts. We used two experimental approaches because the nuclear matrix protein composition depends largely on the isolation conditions, making its definition mostly operational. In situ studies and in vitro experiments performed in 1361.5 cells, a cell line in which multiple copies of the construct have been established, indicate that two interesting regions of the construct interact with the nuclear matrix. The first region is located in the v-Ha-ras gene 5'-flanking sequences. These sequences come from the Harvey virus and contain a piece of the virus like 30S (VL30) sequences in which the v-Ha-ras gene is embedded. This DNA fragment was coupled to the thymidine kinase (TK) promoter driving the reporter luciferase gene and assayed in transient transfection experiments. Its insertion, in the sense orientation, upstream of the TK promoter resulted in a moderate enhancement (2-3-fold) of the luciferase activity. The second region is the most interesting from a physiological point of view. It contains the plasmid maintenance sequence 1 (PMS-1) and the core origin of replication of the bovine papilloma virus. Differences in the results from in situ (nuclear scaffold) and in vitro (nuclear matrix) experiments suggest that the components involved in the interaction with PMS-1 and the viral origin of replication are different. This may be of importance in the context of the recently proposed view that PMS-1 could be part of a composite origin of replication and provide information at a distance.

3T3 Cells↗
PubMed1988

Sequence and structural requirements of a herpes simplex viral DNA replication origin.

Herpes simplex virus (HSV) types 1 and 2 contain two classes of origins of DNA replication, oriS and oriL, which are closely related. A series of plasmids was constructed which contained specifically altered versions of the HSV type 2 oriS replication origin. Their ability to replicate in an in vivo replicon assay allowed a core origin of 75 base pairs (bp) to be defined. It included both arms of a 56-bp palindrome and from 13 to 20 bp of sequence leftward of the palindrome. The AT-rich sequence at the center of the palindrome was essential. Sequences on either side of the core origin enhanced replication. When additional copies of the -AT-dinucleotide were introduced progressively into the center of the palindrome, an oscillating effect on origin function was observed. These and other data implicate a linear rather than a cruciform conformation of the oriS palindrome in the initiation of HSV replication.

Base Sequence↗
PubMed1978

Mini-chromosomes: plasmids which carry the E. coli replication origin.

We have isolated plasmids by linking the 5.9 MD EcoRI fragment of E. coli that carries the origin of replication to an EcoRI fragment that carries an amplicillin resistance determinant, but lacks an origin of replication. 3 plasmids of this type, pOC1, pOC2, and pOC3, are described in detail in this report. Although the plasmids have some adverse effect on the growth properties of the host strain, their existence shows that two functioning chromosomal origins can coexist in one cell. Deletions generated from this type of plasmids allow an allocation of the origin of replication of E. coli within a DNA segment less than 0.4 MD in size.

Ampicillin↗
PubMed1989

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.

Amino Acid Sequence↗
PubMed1992

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.

Amino Acid Sequence↗
PubMed1989

Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication.

Replication of the chromosome of bacteriophage lambda depends on the cooperative action of two phage-coded proteins and seven replication and heat shock proteins from its Escherichia coli host. As previously described, the first stage in this process is the binding of multiple copies of the lambda O initiator to the lambda replication origin (ori lambda) to form the nucleosomelike O-some. The O-some serves to localize subsequent protein-protein and protein-DNA interactions involved in the initiation of lambda DNA replication to ori lambda. To study these interactions, we have developed a sensitive immunoblotting protocol that permits the protein constituents of complex nucleoprotein structures to be identified. Using this approach, we have defined a series of sequential protein assembly and protein disassembly events that occur at ori lambda during the initiation of lambda DNA replication. A second-stage ori lambda.O (lambda O protein).P (lambda P protein).DnaB nucleoprotein structure is formed when O, P, and E. coli DnaB helicase are incubated with ori lambda DNA. In a third-stage reaction the E. coli DnaJ heat shock protein specifically binds to the second-stage structure to form an ori lambda.O.P.DnaB.DnaJ complex. Each of the nucleoprotein structures formed in the first three stages was isolated and shown to be a physiological intermediate in the initiation of lambda DNA replication. The E. coli DnaK heat shock protein can bind to any of these early stage nucleoprotein structures, and in a fourth-stage reaction a complete ori lambda.O.P.DnaB.DnaJ.DnaK initiation complex is assembled. Addition of ATP to the reaction enables the DnaK and DnaJ heat shock proteins to mediate a partial disassembly of the fourth-stage complex. These protein disassembly reactions activate the intrinsic helicase activity of DnaB and result in localized unwinding of the ori lambda template. The protein disassembly reactions are described in the accompanying articles.

Bacteriophage lambda↗
PubMed1987

Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.

DNA Helicases↗
PubMed1985

Conformational changes in a replication origin induced by an initiator protein.

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.

Bacterial Proteins↗
PubMed1990

Alpha and beta replication origins of plasmid R6K show similar distortions of the DNA helix in vivo.

Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.

Base Sequence↗
PubMed1977

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.

DNA Replication↗
PubMed1974

More precise mapping of the replication origin in Escherichia coli K-12.

The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for lambdaind(-) and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, lambda, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.

Amino Acids↗
PubMed1972

Deoxyribonucleic acid-deoxyribonucleic acid hybridization assay for replication origin deoxyribonucleic acid of Escherichia coli.

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.

Amino Acid Sequence↗
PubMed1986

A replication origin is turned off by an origin-"silencer" sequence.

The chromosome of R6K contains multiple origins of replication. The origin gamma is infrequently used in the original plasmid and remains "silent" in certain miniplasmid derivatives. The inactivation of the origin is caused by a natural origin silencer located adjacent to the minimal ori gamma sequence. The silencer functions in cis and has no trans activity. It has functional polarity and works only in one orientation when present immediately downstream from ori gamma. The silencer apparently initiates an RNA that invades ori gamma and turns it off either by competing with a primer RNA or by disrupting ori gamma structure. As predicted, removal of the silencer blocks the synthesis of silencer RNA and derepresses the origin.

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↗