Functional analysis of minichromosome replication: bidirectional and unidirectional replication from the Escherichia coli replication origin, oriC.

Replicating molecules of minichromosomes pCM959 and pOC24 were analyzed by electron microscopy. Replication of pCM959 proceeded bidirectionally from the replication origin, oriC, in about 60% of the molecules; the rest of the molecules replicated unidirectionally in either direction. pOC24, in which deoxyribonucleic acid to the right (clockwise) of the oriC segment is deleted, seemed to replicate predominantly unidirectionally counterclockwise from oriC.     

Asymmetric replication of an oriC plasmid in Escherichia coli

Summary Plasmid pTSO118 containing the Escherichia coli origin of replication, oriC, initiated replication simultaneously with the chromosome when temperature-sensitive host cells were synchronized by temperature shifts. Replicating intermediates of the plasmid as well as of the chromosome were isolated from the outer membrane fraction of the cell. Plasmid DNA with eye structures was enriched when cytosine-1--arabinofuranoside was introduced into the culture during replication. Electron microscopy of the replicating molecules, after digestion with restriction endonucleases, showed that the replication fork proceeds exclusively counter-clockwise towards the unc operon. We conclude that the replication of the oriC plasmid is unidirectional or, if bidirectional, is highly asymmetric.     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

The structure of the initiation complex at the replication origin, oriC, of Escherichia coli.

Two distinct regions in the replication origin, oriC, of Escherichia coli are separately distorted upon initiation complex formation by the initiator protein DnaA. The AT-rich region in the left part of oriC and the start site region in the right part of oriC. Chemical modification of single-stranded DNA was observed at both regions whereas endonuclease recognition of DNA mini-bulges specifically occurred in the start site region. We show that the helical phasing of binding sites for DnaA protein in oriC is important for origin function. An insertion or deletion of one helical turn between the two rightmost binding sites does not alter the efficiency of replication initiation, whereas all modifications of distance by less or more than one helical turn result in inactivation of oriC. DnaA binding and helical distortions in the AT-rich region as well as in the start site region are not affected in the distance mutants irrespective of their functionality in vivo. We propose a specific compact nucleoprotein structure for the initiation complex.     

Start sites for bidirectional in vitro DNA replication inside the replication origin, oriC, of Escherichia coli.

In vitro replication of mini-chromosomes in the absence of DNA ligase activity resulted in replication products with single-strand breaks at specific sites. The occurrence of these nicks was coupled to an active replication process, therefore we expect them to represent start sites for DNA replication. Two positions within oriC for each of the two leading strands of bidirectional replication were found. Within each position are one or two start sites. Counterclockwise synthesis started at positions 194/199 and 265/272, clockwise synthesis at positions 209/219 and 254. The start positions are located close to DnaA protein binding sites. A model for initiation accommodating this observation is discussed.     

A temperature upshift induces initiation of replication at oriC on the Escherichia coli chromosome

A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat-induced replication (HIR) initiates at oriC , is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41°C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.     

The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro.

Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.     

Overinitiation of replication of the Escherichia coli chromosome from an integrated runaway-replication derivative of plasmid R1.

A 16-base-pair fragment, deletion of which completely inactivated oriC, was replaced by a temperature-dependent runaway-replication derivative (the copy number of which increases with temperature) of the IncFII plasmid R1. The constructed strains were temperature sensitive, and flow cytometry revealed a severalfold increase in the DNA/mass ratio following shifts to nonpermissive temperatures. The cell size distribution was broader in the constructed strains relative to that in the wild type because of asynchrony between the chromosome replication and cell division cycles. This difference was more pronounced for counterclockwise initiation of chromosomal replication, in which small DNA-less cells and long filaments were abundant. Following a temperature shift the cell size distributions became even more broad, showing that changes in the frequency of chromosomal replication affect cell division and emphasizing the interplay between these two processes.     

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.     

The DnaA box R4 in the minimal oriC is dispensable for initiation of Escherichia coli chromosome replication.

We have developed a genetic system with which to replace oriC+ on the Escherichia coli chromosome with modified oriC sequences constructed on plasmids. Using this system we have demonstrated that chromosomal oriC can tolerate the insertion of a 2 kb fragment at the HindIII site between DnaA boxes R3 and R4, whereas the same insertion completely inactivates cloned oriC. We have further found that although R4 is essential for the origin activity of cloned oriC, cells carrying a deletion of R4 in chromosomal oriC are viable. These results indicate that the oriC sequence necessary for initiation of chromosome replication is different from the so-called minimal oriC that was determined with cloned oriC. Flow cytometric analyses have revealed that these oriC mutations confer the initiation asynchrony phenotype. Introduction of the R4 deletion into a fis::kan mutant, which lacks the DNA bending protein FIS, renders the mutant cells inviable.     

SeqA limits DnaA activity in replication from oriC in Escherichia coli

A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated. Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles. The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold). The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.     

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.     

Random replication of the stringent plasmid R1 in Escherichia coli K-12.

The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication). The replication of R1 was studied in a density-shift experiment. One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice. The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication. This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication). Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication. However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle. The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed.     

Replication initiation at the Escherichia coli chromosomal origin

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.     

Replisome assembly at oriC, the replication origin of E. coli, reveals an explanation for initiation sites outside an origin.

This study outlines the events downstream of origin unwinding by DnaA, leading to assembly of two replication forks at the E. coli origin, oriC. We show that two hexamers of DnaB assemble onto the opposing strands of the resulting bubble, expanding it further, yet helicase action is not required. Primase cannot act until the helicases move 65 nucleotides or more. Once primers are formed, two molecules of the large DNA polymerase III holoenzyme machinery assemble into the bubble, forming two replication forks. Primer locations are heterogeneous; some are even outside oriC. This observation generalizes to many systems, prokaryotic and eukaryotic. Heterogeneous initiation sites are likely explained by primase functioning with a moving helicase target.     

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.