Map position of the replication origin on the E. coli chromosome.

Summary Strains carrying a dnaA temperature sensitive (t.s.) mutation and a Mu-1 prophage inserted within different genes near the origin of replication have been constructed. For each strain, integratively suppressed Hfrs, named G and D, in which the ori region was replicated clockwise and counterclockwise respectively, were isolated. The strand preferences of Mu-1 specific Okazaki fragments were subsequently determined for each t.s. strain and its Hfr derivatives. Their comparison led us to establish the direction of replication of the Mu-1 marker from ori. The site ori was thus confined to the bglB-C-rbsK-P interval.     

Location and characterisation of a new replication origin in the E. coli K12 chromosome.

Summary A segment of DNA located in the region of the E. coli K12 chromosome previously identified by the Rac phenotype can function as a self-replicating plasmid. Evidence is presented that this plasmid, the oriJ plasmid, contains the origin of replication of a defective prophage postulated to be located in this chromosomal region by Low (1973). The plasmid can only be maintained in strains in which this postulated prophage has been deleted. In strains which possess the prophage selection for plasmid maintenance permits the isolation of clones containing new deletions which we postulate are the result of prophage excision.     

On the origin of replication of Escherichia coli chromosome

DNA labelled with [³H]thymine near the beginning or near the terminus of chromosomal replication was hybridized with isolated F′13, F′14 and F′15 DNA's. The presented results show that the ilv marker replicates earlier than the lac or thy markers in the cycle of chromosomal replication of Escherichia coli strains, K12F⁻ AY5 and 15T⁻ 557.     

ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome

ATP is bound to dnaA protein with high affinity (KD = 0.03 microM) and hydrolyzed slowly to ADP in the presence of DNA. ADP is also bound tightly to dnaA protein and exchanges with ATP very slowly. The ATP form is active in replication; the ADP form is not. A unique conformation of oriC, formed in an early initiation stage, depends on dnaA protein being in the ATP form. The subsequent entry of dnaB protein to form a prepriming complex also requires ATP binding and is blocked by bound ADP. Inasmuch as hydrolysis of ATP is far slower than these initiation reactions and since the poorly hydrolyzable analogue ATP gamma S can replace ATP, the ATP function appears to be allosteric. The extraordinary affinity of ATP for dnaA protein, its slow hydrolysis to ADP, the profound inhibition of dnaA functions by ADP, and the very slow exchange of ADP all point to a possible regulatory role for these nucleotides in the cell cycle.     

Insertion of an R1 plasmid into the origin of replication of the E. coli chromosome: random timing of replication of the hybrid chromosome

A 16 bp BgI II fragment was deleted in vitro from the minimal origin of replication of the Escherichia coli chromosome, oriC, and was replaced by a 10 kb R1 miniplasmid, pKN1562, containing the basic R1 replicon and a kanamycin resistance gene. The deletion-insertion was transferred by homologous recombination into the chromosome of a dnaA(ts) strain. P1 transduction separated the origin "mutation" from the dnaA46 allele. Integration of mini-R1 into oriC was verified by Southern blotting and by analysis of the R1 incompatibility phenotype. It was possible to isolate normal R1 miniplasmids from the integrated R1. Chromosome replication was initiated at random times after a short delay. The constructed strains grew 20%-30% slower than the wild type and showed more heterogeneous cell sizes.     

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

Summary 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 ampicillin 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.     

oriX: A new replication origin in E. coli

Replication of the chromosome of E. coli at 42°C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42°C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxal mutant of one of these Hfrs, we have shown that replication proceeds at 42°C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.     

On the localization of the origin of DNA replication in E. coli.

A theory is presented to describe the behavior of micro-organisms, bacteria and protozoa. Individual cells are regarded as particles having internal state variables. The change of each variable with time depends on the environmental condition. The velocity and the frequency of direction change of swimming cells are determined by the values of these variables. With this framework, the theory gives a method to connect the behaviour in a spatial gradient of the environment and the behaviour upon a change of the environment with time. Observed behaviors of bacteria and protozoa are understandable on the basis of simple rate equations for internal state variables and the product expressions for the velocity and the frequency of direction change as functions of these variables. Experimental data on the thermotaxis of paramecium are shown for comparison with the theoretical results.     

Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin

Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase-dependent. As a major component of the bacterial nucleoid, HU binds to DNA non-specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the alpha dimer of HU is more active than other forms of HU in initiation of an oriC-containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the alpha subunit of HU interacts with the N-terminal region of DnaA. These observations support a model whereby DnaA interacts with the alpha dimer or the alphabeta heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the alpha dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the alphabeta heterodimer or the beta dimer.     

Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome

Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This "open complex" requires ATP and 38 degrees C for optimum formation and stability. The subsequent dnaC-dependent entry of dnaB helicase to form a "prepriming complex" stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication.     

Sequence organization of replication origin of the Escherichia coli K-12 chromosome

A sequence of 245 base-pairs (oriC) in the replication origin of the Escherichia coli K-12 chromosome has been shown to provide all the information essential for initiation of bidirectional replication. In order to elucidate the sequence organization of oriC, numerous mutants carrying a single-to-multiple transitions from G X C to A X T base-pair were constructed by localized mutagenesis in vitro, which uses sodium bisulfite, and the correlation between the mutation sites and replicating ability (Ori function) was systematically analyzed. By isolating non-defective (Ori+) mutants with multiple base changes, transitions at 71 positions among 101 G X C pairs in oriC were found to have no effect on Ori function. Investigation of defective (Ori-) mutants, on the other hand, showed that individual replacements at 18 positions were detrimental to Ori function to some extent. These irreplaceable G X C pairs fell in the positions where no substitution was detected in the Ori+ mutants. The defect of the Ori- mutants with a single base substitution was generally weaker than that of the previously constructed Ori- mutants lacking a part of oriC. The addition of two or more base changes each giving a faint Ori- phenotype, however, resulted in a more intensive Ori- phenotype. We have previously demonstrated that oriC contains several regions where deletion or insertion of oligonucleotides leads to strong Ori- phenotypes. Transitions in those areas did not cause any defect of Ori function. Combining present results on base substitution mutants with the previous observations together, we assumed that the oriC sequence provides multiple interaction sites with replication initiation factors, and the precise arrangement of these sites are required for Ori function.     

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.     

In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication.

Summary The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.