The segregation of Escherichia coli minichromosomes constructed in vivo by recombineering

Circularized regions of the chromosome containing the origin of replication, oriC, can be maintained as autonomous minichromosomes, oriC plasmids. We show that oriC plasmids containing precise, pre-determined segments of the chromosome can be generated by a simple in vivo recombineering technique. We generated two such plasmids carrying fluorescent markers. These were transferred to a recipient strain with a different fluorescent marker near the chromosomal copy of oriC. Thus the fates of the oriC plasmid and chromosomal origins could be followed independently in living cells by fluorescence microscopy. In contrast to a previous report, we show that there is a strong tendency of oriC plasmid copies to accumulate at the cell center as a single or double focus at the plane of cell division. This is not simply due to exclusion from the nucleoid space but rather appears to be a specific recognition and retention of the plasmid by some central-located cell site.     

The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin

Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.     

Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system

Fluorescence in situ hybridization (FISH) analysis has revealed the subcellular localization of specific chromosomal segments and plasmid molecules during the cell division cycle in Escherichia coli: the replication origin (oriC) segments on the chromosome are localized at nucleoid borders, and actively partitioning mini-F plasmid molecules are localized at the 1/4 and 3/4 positions of the cell. In contrast, mini-F plasmid molecules lacking the sopABC segment are randomly localized in cytoplasmic areas at cell poles. In this study, we analysed the subcellular localization of an oriC plasmid that contains the minimum E. coli chromosomal replication origin and its flanking regions. These oriC plasmid molecules were mainly localized in cytosolic areas at cell poles. On the other hand, oriC plasmid DNA molecules carrying the sopABC segment of F plasmid were localized at cell quarter sites, as were actively partitioning mini-F plasmid DNA molecules. Therefore, we conclude that oriC itself and its flanking regions are not sufficient for positioning the replication origin domain of the E. coli chromosome within the cell.     

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.     

The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi-forked replication

Replication initiation is a key event in the cell cycle of all organisms and oriC , the replication origin in Escherichia coli , serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriC s for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.     

Similarities in control of mini-F plasmid and chromosomal replication in Escherichia coli.

In Escherichia coli, concentrations of a mini-F plasmid with two origins of replication (oriV and oriS) were 50% lower in fast-growing cells than in slow-growing cells. By contrast, a mini-F plasmid deleted for oriV maintained a uniform concentration in both fast- and slow-growing cells, and in this behavior the plasmid mimicked the control by the host of chromosomal origin (oriC) concentration.     

The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin.

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.     

Replication of M13 oriC bacteriophages in Escherichia coli rep mutant is dependent on the cloned Escherichia coli replication origin.

The involvement of the Escherichia coli rep protein in the replication of M13 chimeric deoxyribonucleic acids (DNAs) carrying the E. coli chromosomal DNA replication origin (oriC) has been examined. Previous studies indicate that the cloning of a 3,550-base-pair sequence of chromosomal DNA containing oriC into an M13 vector allows extensive replication of the M13 oriC chimeric DNA in an E. coli rep-3 mutant. We have extended these studies by preparing a 330-base-pair deletion that specifically deletes the oriC sequence in the M13 oriC DNAs, to demonstrate that the replication observed in the rep-3 host is dependent on the cloned origin. Thus, a DNA-unwinding enzyme other than the rep protein may be involved in the strand separation process accompanying replication which initiates at oriC in the M13 oriC chimeric DNAs and in the E. coli chromosome. The rep assay used for assessing the functionality of the cloned oriC is useful for analysis of any rep-independent origin of replication functional in E. coli. A direct selection for a cloned origin of replication is possible in the rep-3 recA56 host. Since the cloned origin is nonessential for propagation of the M13 chimeric phage in a rep+ host, mutations in the cloned origin may be constructed, and the mutant phage may be examined by a simple transductional analysis of the rep-3 recA56 mutant strain.     

Novel Escherichia coli mutant, dnaR, thermosensitive in initiation of chromosome replication.

A newly isolated Escherichia coli mutant thermosensitive in DNA synthesis had an allele named dnaR130, which was located at 26.3 minutes on the genetic map. The mutant was defective in initiation of chromosome replication but not in propagation at a high temperature. This mutant was capable of growing in the absence of the rnh function at the high temperature by means of a dnaA-independent replication mechanism. In the mutant exposed to the high temperature, an oriC plasmid was able to replicate, although at a lower rate than at the low temperature. The plasmid replication at the high temperature depended on the dnaA function essential for the initiation of replication from oriC. The mutant lacking the rnh function persistently maintained the oriC plasmid at the high temperature in a dnaA-dependent manner. Thus, the dnaR function was required for initiation of replication of the bacterial chromosome from oriC but not the oriC plasmid. This result reveals that a dnaR-dependent initiation mechanism that is dispensable for oriC plasmid replication operates in the bacterial chromosome replication.     

DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.     

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.     

Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III

Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria. The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity. This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein. Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2. DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV. The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E. coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors. Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands. These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.     

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.     

The mutH gene regulates the replication and methylation of the pMB1 origin.

DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColE1 origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMB1 origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMB1 replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.     

Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells.

Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed. Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome. The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC. Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact and functional oriC sequence. The seqA2 mutation was found to overcome the incompatability phenotype by increasing the cellular oriC copy number 3-fold thereby allowing minichromosomes to coexist with the chromosome. The replication pattern of a wild-type strain with multiple integrated minichromosomes in the oriC region of the chromosome, led to the conclusion that initiation of DNA replication commences at a fixed cell mass, irrespective of the number of origins contained on the chromosome.     

Stoichiometry of DnaA and DnaB protein in initiation at the Escherichia coli chromosomal origin

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.