Origin and sequence of chromosome replication in Escherichia coli

Two methods have been used to determine the origin and direction of chromosome replication in Escherichia coli: gradient of marker frequency and sequence of replication in synchronized cultures. In both cases, DNA-DNA hybridization was used to assay for gene dosage. A series of isogenic strains were made lysogenic for phage λ and for phage Mu-1, with phage Mu-1 in a different chromosomal location in each strain. In a first group of experiments, DNA from exponential cultures of the various strains was extracted, denatured, immobilized on filters and hybridized against a mixture of differentially labeled phage λ and phage Mu-1 DNA. This was done for several culture conditions. The ratio of hybridization Mu-1/λ gives a measurement of the dosage of the chromosome region where phage Mu-1 is integrated. A plot of this ratio versus map position reflects the marker frequency distribution.     

An origin of DNA replication from streptomycete phage ΦU1

A DNA fragment from phage ΦU1 containing an origin of DNA replication was identified. This fragment, designatedori, was able to support the maintenance inStreptomyces lividans of a plasmid lacking a functional Gram-positiveori. The sequence of the minimalori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in thisori fragment indicates that host factors alone were sufficient to initiate replication atori.     

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.     

A comparative study of the ori sequences from the mitochondrial genomes of twenty wild-type yeast strains

The ori sequences of the mitochondrial genomes of 20 wild-type strains of Saccharomyces cerevisiae were compared with those of the previously studied strain A (de Zamaroczy et al., 1984). The seven canonical ori sequences of this strain appear to be present in all strains tested, but in most strains ori1 is replaced by an extensively rearranged ori1 ¹ sequence, and an additional ori sequence, ori8, is present between the oxi3 and the 15S RNA genes; one strain, B, lacks ori4. The location and orientation of ori sequences of three strains, B, C and K, were found to be the same as in strain A. The primary structures of four ori sequences from three different strains (ori1 of strain J69-1B, ori3 and ori5 of strain K, ori6 of strain D273-10B) were found to be identical with the corresponding ori sequences previously investigated. Hybridization experiments with different on probes indicated a conservation of ori2–ori7 sequences in all strains tested. The primary structure of a petite genome derived from strain B and carrying ori1 ¹ is reported and discussed.     

Differential use of multiple replication origins in the ribosomal DNA episome of the protozoan parasite Entamoeba histolytica

The factors that control the initiation of eukaryotic DNA replication from defined origins (oris) on the chromosome remain incompletely resolved. Here we show that the circular rDNA episome of the human pathogen Entamoeba histolytica contains multiple potential oris, which are utilized in a differential manner. The primary ori in exponentially growing cells was mapped close to the promoter of rRNA genes in the upstream intergenic spacer (IGS) by two-dimensional gel electrophoresis. Replication initiated predominantly from the upstream IGS and terminated in the downstream IGS. However, when serum-starved cells were allowed to resume growth, the early oris which became activated were located in other parts of the molecule. Later the ori in the upstream IGS became activated, with concomitant silencing of the early oris. When the upstream IGS was located ectopically in an artificial plasmid, it again lost ori activity, while other parts of the rDNA episome could function as oris in this system. Therefore, the activation or silencing of the ori in this episome is context dependent, as is also the case with many eukaryotic replicons. This is the first replication origin to be mapped in this primitive protozoan and will provide an opportunity to define the factors involved in differential ori activity, and their comparison with metazoans.     

Characterization of the Pea Chloroplast DNA OriA Region

One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the ori A region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3′ end of the 16S rRNA gene and the Eco RI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the Bam HI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from ori A. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dna A-like binding sites, most of which are unique to the pea ctDNA ori A region when compared with published rRNA spacer sequences from other chloroplast genomes.     

Sequence rearrangements at theori 7 region ofSaccharomyces cerevisiae mitochondrial DNA

Summary Threeori elements (ori 2,ori 5, andori 7) have been sequenced inSaccharomyces cerevisiae strain Dip 2 and compared to the equivalentori elements of a second strain (B). Bothori 2 andori 5 exhibit 98% base matching between strains Dip 2 and B. In contrast, the thirdori element (ori 7) exhibits extensive sequence rearrangements whereby a segment located downstream in the consensus strain occurs within theori structure in Dip 2. This represents a novel polymorphic form of the yeast mitochondrial genome.     

Nuclear‐encoded DnaJ homologue of Plasmodium falciparum interacts with replication ori of the apicoplast genome

The apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via a d ‐loop/bi‐directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA–protein interactions at a plDNA replication ori region and report the identification of a nuclear‐encoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJ_A interacted with the minor groove of the DNA double‐helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti‐PfDnaJ_A antibodies confirmed identity of the protein in DNA‐binding experiments with organellar protein fractions. The DNA‐binding domain of the ～69 kDa PfDnaJ_A lay within the N‐terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJ_A in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non‐specific manner. Our results suggest a role for PfDnaJ_A in replication/repair of the apicoplast genome.     

Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2

Summary Replication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small ori _RK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus incompatible with RK2 replicons. However, the 393 bp ori _RK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on ori _RK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII ori_RK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from ori _RK2is required or may simply reflect the strong selective pressure for establishment of the incoming ori _RK2 plasmid and poor ability of the HpaII ori _RK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII ori _RK2 may be designated inc ₁. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc ₂ region, to distinguish it from inc ₁, above, that overlaps but does not coincide with the 393 bp HpaII ori _RK2. This inc ₂ region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete ori _RK2. Also, only part of the inc ₂ region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for ori _RK2.     

Nucleotide Sequence and Genetic Characterization of the Novel IncQ-like Plasmid pIE1107

The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids. Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems. However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons. Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons. Experiments with clonedoriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its ownoriV which differs only by a few nucleotides from theoriV of IncQ plasmids. Implications from the apparent highly specific protein–DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed.     

Chromosome mobilization and integration of F-factors in the chromosome of RecA strains of E. coli under the influence of bacteriophage Mu-1

Summary Transfer of chromosome promoted by an F⁺ or an F needs a recombinational event between episome and chromosome and is therefore very low in RecA strains (Wilkins, 1969).When F⁺ or F RecA cells are mated with F^- cells and simultaneously infected with bacteriophage Mu-1 the transfer of chromosome is greatly stimulated and seems to take place on any site of the chromosome even when homology is present between the F and the chromosome.A spot-test based on Mu-1 promoted chromosome-mobilization was developed to search for mutants of Mu-1 that had lost the ability to promote chromosome-mobilization. Two conditional lethal amber mutants falling in different complementation groups were found to have lost this property.The integration of an Ftslac ⁺ in the chromosome of a RecA strain is also strongly stimulated by Mu-1 and not site-specific. The resulting Hfr's are very stable and of the clockwise and counterclockwise types. Some of the integrated F-primes are sterile and not able to transfer chromosome or do not form F-pilli.Also the Mu-1 stimulated integration of an Fts(nadA-chlA)⁺ in the chromosome of a RecA strain which has a deletion on the chromosome from nadA-chlA was studied. It was found that when the F is integrated under the influence of Mu-1 also episomal genes carried by that F can be inactivated.     

Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species

A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis.     

The atypical response regulator HP1021 controls formation of the Helicobacter pylori replication initiation complex

The replication of a bacterial chromosome is initiated by the DnaA protein, which binds to the specific chromosomal region oriC and unwinds duplex DNA within the DNA‐unwinding element (DUE). The initiation is tightly regulated by many factors, which control either DnaA or oriC activity and ensure that the chromosome is duplicated only when the conditions favor the survival of daughter cells. The factors controlling oriC activity often belong to the protein families of two‐component systems. Here, we found that Helicobacter pylori oriC activity is controlled by HP1021, a member of the atypical response regulator family. HP1021 protein specifically interacts with H. pylori oriC at HP1021 boxes (5′‐TGTT[TA]C[TA]‐3′), which overlap with three modules important for oriC function: DnaA boxes, the hypersensitivity (hs) region and the DUE. Consequently, HP1021 binding to oriC precludes DnaA‐oriC interactions and inhibits DNA unwinding at the DUE. Thus, HP1021 constitutes a negative regulator of the H. pylori orisome assembly in vitro. Furthermore, HP1021 boxes were found upstream of at least 70 genes, including those encoding CagA and Fur proteins. We postulate that HP1021 might coordinate chromosome replication, and thus bacterial growth, with other cellular processes and conditions in the human stomach.     

Vegetative recombination of bacteriophage Mu-1 in Escherichia coli

Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec⁺ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination.The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.Research Fellow from the Laboratory of Genetics, State University, Leiden, The Netherlands.     

Mycobacterium tuberculosis oriC sequestration by MtrA response regulator

The regulators of Mycobacterium tuberculosis DNA replication are largely unknown. Here, we demonstrate that in synchronously replicating M. tuberculosis, MtrA access to origin of replication (oriC) is enriched in the post‐replication (D) period. The increased oriC binding results from elevated MtrA phosphorylation (MtrA～P) as evidenced by reduced expression of dnaN, dnaA and increased expression of select cell division targets. Overproduction of gain‐of‐function MtrA_Y102C advanced the MtrA oriC access to the C period, reduced dnaA and dnaN expression, interfered with replication synchrony and compromised cell division. Overproduction of wild‐type (MtrA+) or phosphorylation‐defective MtrA_D56N did not promote oriC access in the C period, nor affected cell cycle progression. MtrA interacts with DnaA signaling a possibility that DnaA helps load MtrA on oriC. Therefore, oriC sequestration by MtrA～P in the D period may normally serve to prevent untimely initiations and that DnaA–MtrA interactions may facilitate regulated oriC replication. Finally, despite the near sequence identity of MtrA in M. smegmatis and M. tuberculosis, the M. smegmatis oriC is not MtrA‐target. We conclude that M. tuberculosis oriC has evolved to be regulated by MtrA and that cell cycle progression in this organisms are governed, at least in part, by oscillations in the MtrA～P levels.     

Replication origin of the Escherichia coli K-12 chromosome: The size and structure of the minimum DNA segment carrying the information for autonomous replication

Summary A DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA ⁺ cells, whereas pBR322 replicates only in polA ⁺ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori⁺ and Ori^- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure.To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori^- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.     

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: involvement of host cellular factors

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.     

Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

It has previously been concluded that regions tentatively designated trfA and trfB, located at 16–18.7 and 54–56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, ori_RK2, located at 12 kb on the genome, is also required. A segment of DNA containing ori_RK2 can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans-acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trfA region alone can provide the trans-acting functions necessary for replication from ori_RK2. Deletion of the trfB region in trans to an ori_RK2 plasmid does not correlate with alteration in copy number or stability of the ori_RK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trfA and trfB regions as indicated by complementation analysis of three different mutants. The trfA and trfB regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent ori_RK2 plasmid to replicate at 30 °C. When the source of trfA and trfB is a trfB mutant the ori_RK2 plasmid is temperature stable but is temperature sensitive when the source is a trfA mutant. This confirms that only trfA is essential for initiation at and elongation from ori_RK2 which is probably the primary event in RK2 replication and suggests that the trfB region plays some other role in plasmid maintenance in plasmids carrying all three regions, ori_RK2, trfA, and trfB.     

Straight core structure of DNA replication origins in the mammalian AMPD2 locus

Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (～300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.