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PubMed1989

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221.

pT181 and pC221 are closely related Staphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in the rep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping the rep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1981

The remarkable instability of replication loops provides a general method for the isolation of origins of DNA replication.

Closed superhelical replicative intermediates of SV40 DNA were stable during incubation at elevated temperatures for prolonged times. However, when the replicative intermediates were nicked or made linear by digestion either outside or inside of the replication loop, the nascent DNA strands were spontaneously extruded at a measurable rate even at 37 degrees C. The extruded DNA was double-stranded. The rate-limiting step in the extrusion was not the rate of branch migration. The instability of replication loops suggested a method for the isolation of replication origins. The method has been tested by a reconstruction experiment in which a small amount of linear SV40 replicative intermediates was mixed with a vast excess of cellular DNA. The mixture was heated overnight at 55 degrees C, and the spontaneously extruded nascent strands were isolated and inserted into pBR322. Plasmids containing SV40 DNA of less than genomic size, symmetrically distributed about the SV40 replication origin, were isolated. This result suggests that the method is generally applicable to the isolation of replication origins.

DNA Replication↗
PubMed1969

Origin and sequence of chromosome replication in Neisseria meningitidis: influence of a genetic factor determining competence.

Chromosome replication in Neisseria meningitidis was examined by enumerating mutants induced by nitrosoguanidine during synchronous replication after release from prolonged chloramphenicol inhibition. Clear maxima of mutagenesis were observed at certain times. These times were different for seven individual markers. At a definite time after the first maximum, there was a second one. The intervals between successive maxima were identical for all markers in each experiment. This permitted the construction of replication maps which were in general agreement with mapping based on marker frequency analysis by use of a transformation system. Comparison of replication in genetically competent and genetically incompetent variants substantiated the previous assumption that the change from genetic competence to genetic incompetence in this strain results in a change in the replication origin as well as in the direction of replication.

Chromosomes, Bacterial↗
PubMed1996

Increased transcomplementation properties of plasmids carrying HSV-1 origin of replication and packaging signals.

This report describes a very simple method that facilitates the analysis of the functional activity of essential genes of herpes simplex virus type 1 (HSV-1) in cell culture. The method, which depends on the use of complementing plasmids containing a virus origin of replication (ori s) and a packaging signal (amplicon plasmids), was tested for its ability to detect complementation of the defective HSV-1 Cgal delta 42 virus, a UL42 null mutant, by amplicon plasmids carrying either wild-type (pA-UL42) or mutated alleles (pA-delta UL42) of the HSV-1 UL42 gene. In nonpermissive Vero cells transfected with amplicon plasmid pA-UL42 and superinfected with the defective Cgal delta 42 virus, both the plasmid and the helper genomes were amplified and packaged, giving rise to a self-complementing amplicon/helper virus population able to disseminate and to form lytic plaques in cell culture. These plaques were due to complementation and not to recombination between the defective virus and the amplicon plasmid as confirmed by Southern blot analysis of individual plaques. No complementation was observed by superinfection of cells transfected with the noncomplementing pA-delta UL42 amplicon plasmid. Instead, small foci were observed in cells transfected with plasmids which express wild-type UL42 but were unable to amplify or to become packaged, and the few true lytic plaques that were observed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complementation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess function activity of an HSV-1 essential gene in a one-step experiment and (ii) oris-carrying plasmids could be instrumental in the production and selection of recombinant HSV-1 vectors.

Animals↗
PubMed2000

Multiple deletions of mtDNA remove the light strand origin of replication.

Idiopathic inflammatory myopathies are progressive, debilitating muscle diseases. The pathogenesis of these disorders is multifactorial and appears to include mutations of the mitochondrial genome, which are usually indicated by morphological changes of mitochondria. The vast majority of all mitochondrial DNA deletions found are located between the origins of replication in the "major region" between nt5760-nt190. Using long distance PCR and sequencing techniques, we detected deletions which were unusually large (ca. 10500-12800 bp) and show uncommon 5'-breakpoints between nt800 and nt3326. Unlike most other deletions, their breakpoints are far upstream of the "major region." The atypical location of these deletions suggests a different pathomechanism. The impact of the mitochondrial DNA deletions in the pathogenetic cascade remains uncertain.

Aged↗
PubMed1986

The relationship between chromosomal origins of replication and the nuclear matrix during the cell cycle.

A cytological investigation into the dynamic behaviour of the origins of replication with respect to the nuclear matrix has been carried out on Xenopus laevis cultured cells. In order to preferentially label origins or 'non-origin' regions along DNA fibres, 5-fluoro-2'-deoxyuridine (FUdR)-treated cells were pulsed with [3H]deoxyadenosine in early or late S phase. Samples were then allowed to proceed through the cell cycle for increasing times. The DNA loops were induced in situ to completely uncoil around the nuclear matrix. The autoradiographic analysis shows that, under the experimental conditions used, 'non-origin' regions behave as expected from previous studies, i.e., they associate with the nuclear matrix only when they become part of a replication fork, whereas active origins of replication remain associated with the matrix throughout the cell cycle.

Animals↗
PubMed1984

Expression of a Thiobacillus ferrooxidans origin of replication in Escherichia coli.

A cryptic plasmid from an autotrophically grown arsenic-resistant strain of Thiobacillus ferrooxidans was isolated and cloned into pBR325. The origin of replication of pBR325 was deleted, and the recombinant plasmid was shown to replicate in Escherichia coli, using an origin of replication located on the Thiobacillus plasmid.

Acidithiobacillus thiooxidans↗
PubMed2006

Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins.

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.

Cell Line↗
PubMed1999

Sequence requirements for the assembly of simian virus 40 T antigen and the T-antigen origin binding domain on the viral core origin of replication.

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd(131-260)) have been determined. Binding of the purified T-ag-obd(131-260) is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.

Animals↗
PubMed2006

Evaluation of the lytic origins of replication of Kaposi's sarcoma-associated virus/human herpesvirus 8 in the context of the viral genome.

The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-DeltaOri-R, BAC36-DeltaOri-L, and BAC36-DeltaOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.

Animals↗
PubMed1999

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.

Animals↗
PubMed2003

Sequence diversity and functional conservation of the origin of replication in lactococcal prolate phages.

Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P(E)1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P(E)1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P(E)1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.

Bacteriophages↗
PubMed1979

Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K.

A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and a structural gene for a protein, designated pi, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the pi structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the pi gene. A possible role of the tandem repeats in the regulation of expression of the pi protein and the control of initiation of replication of the plasmid R6K is discussed.

Anti-Bacterial Agents↗
PubMed1997

Binding of the E1 and E2 proteins to the origin of replication of bovine papillomavirus.

DNA replication of bovine papillomavirus (BPV) requires two viral proteins encoded from the E1 and E2 open reading frames. E1 and E2 are sequence-specific DNA binding proteins that bind to their cognate binding sites in the BPV origin of replication (ori). The E1 and E2 proteins can interact physically with each other, and this interaction results in cooperative binding when binding sites for both proteins are present. We have analyzed the binding of E1 to the ori in the absence and presence of E2, using DNase I footprint analysis, gel mobility shift assays, and interference analysis. We have also generated a large number of point mutations in the E1 binding site and tested them for binding of E1 as well as for activity in DNA replication. Our results demonstrate that E1 binds to the ori in different forms in the absence and presence of E2 and that E2 has both a quantitative and a qualitative effect on the binding of E1. Our results also suggest that the ori contains multiple overlapping individual E1 recognition sequences which together constitute the E1 binding site and that different subsets of these recognition sequences are used for binding of E1 in the presence and absence of E2.

Animals↗
PubMed1980

Origin of replication of the DNA of a herpesvirus (pseudorabies).

During the first round of the replication of pseudorabies virus DNA, replicating DNA is mainly in the form of circles. The main origin of replication is located inthe region of the molecule bearing the inverted repeat. Replication proceeds unidirectionally from the origin.

DNA Replication↗
PubMed1985

Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro.

DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma. Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy. Requirement of pi protein for the activity of all these three replication origins in vitro was verified. Frequencies of initiation at the three origins were almost equal.

DNA Replication↗
PubMed1996

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

Bacterial Proteins↗
PubMed1972

Molecular weight of deoxyribonucleic acid synthesized during initiation of chromosome replication in Escherichia coli.

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.

Amino Acids↗