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.     

Feedback controls restrain the initiation of Escherichia coli chromosomal replication

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.     

Regulatory network of the initiation of chromosomal replication in Escherichia coli

The bacterial chromosome is replicated once during the division cycle, a process ensured by the tight regulation of initiation at oriC. In prokaryotes, the initiator protein DnaA plays an essential role at the initiation step, and feedback control is critical in regulating initiation. Three systems have been identified that exert feedback control in Escherichia coli, all of which are necessary for tight strict regulation of the initiation step. In particular, the ATP-dependent control of DnaA activity is essential. A missing link in initiator activity regulation has been identified, facilitating analysis of the reaction mechanism. Furthermore, key components of this regulatory network have also been described. Because the eukaryotic initiator complex, ORC, is also regulated by ATP, the bacterial system provides an important model for understanding initiation in eukaryotes. This review summarizes recent studies on the regulation of initiator activity.     

IciA protein, a specific inhibitor of initiation of Escherichia coli chromosomal replication.

Specific binding of IciA protein to the 13-mers in the origin of a minichromosome (oriC) inhibits initiation of replication in vitro by blocking the opening of this region effected by the initiator DnaA protein (Hwang, D.S., and Kornberg, A. (1990) Cell 63, 325-331). Isolation of the iciA gene (Th?ny, B., Hwang, D.S., Fradkin, L., and Kornberg, A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4066-4070) has made possible the construction of an IciA-overproducing strain, which in turn has simplified the isolation of a large quantity of the protein, indistinguishable from that of the wild-type strain. Based on sedimentation and gel filtration, the IciA protein is an elongated dimer of a 33.4-kDa subunit. The specific binding of IciA protein to the 13-mers was stable for 2 h at 30 degrees C. The amounts of IciA protein, detected by immunoassays, increased 4-fold compared with levels (about 100 dimers) in log-phase cells whereas levels of DnaA protein decreased upon entry of cells into the stationary phase.     

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.     

Strategy for chromosomal gene targeting in RecA-deficient Escherichia coli strains

Reengineering DNA by homologous recombination in Escherichia coli often depends on helper functions provided on a temporarily introduced replicon that is subsequently cured from the cells. The suicide vector pKSS offers a new curing strategy. pKSS specifies a variant of phenylalanyl-transfer RNA (tRNA) synthetase conferring relaxed substrate specificity towards phenylalanine analogs that results in their lethal incorporation into cellular proteins. Consequently, the presence of p-chlorophenylalanine selects for strains that have lost pKSS. This principle, in conjunction with a plasmid-borne recA gene, was exploited for targeted chromosomal mutagenesis by double homologous recombination in RecA-negative E. coli strains. Gene replacement with a kanamycin-resistance cassette was possible in a single step by plating on kanamycin and p-chlorophenylalanine agar plates and incubating at 37 degrees C. The presence of the correct chromosomal mutation and the absence of the plasmid were established by several control experiments. A simple screen confirmed the desired resistance phenotype in 44% of the initially selected clones, and 75% of these had the correct genotype.     

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.     

Isolation and characterization of plasmids carrying a partially defective Escherichia coli replication origin

The replication origin (oriC) of the Escherichia coli chromosome has been cloned and the region essential for chromosomal replication has been delimited to 245 base pairs. In previous studies the ability of recombinants between oriC and ColE1-type vectors, to transform E. coli polA- strains was used to determine which nucleotides in oriC are essential for replication. In this paper we have used a different approach by isolating partial defective replication mutants of a minichromosome (pCM959) that contains oriC as the single replication origin. Our results demonstrate that many mutations are allowed within oriC that do not affect oriC function as measured by the ability to transform E. coli polA- strains. In the minimal oriC region we detected 8 mutations at positions that are conserved in the sequence of six bacterial origins. The implications of these results on previous work will be discussed. Our data also demonstrate that a mutation producing an oriC- phenotype may be suppressed by secondary mutations. An E. coli strain was found that facilitates the isolation of partially defective minichromosomes. The results with this strain indicate a specific function of the sequence surrounding the base pair at position 138.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

Topoisomerase I confers specificity in enzymatic replication of the Escherichia coli chromosomal origin

Crude soluble enzyme fractions that initiate bidirectional replication from the unique Escherichia coli chromosomal origin (oriC) have been fractionated further to identify the components and mechanisms of this complex system. Among the necessary factors is a class of specificity proteins that suppress initiations on plasmids which lack the oriC sequence and which do not depend on dnaA protein. One such specificity factor has been identified as RNase H (Ogawa, T., Pickett, G. G., Kogoma, T., and Kornberg, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1040-1044). Another, described here, has proved to be topoisomerase I. A protein was purified to near homogeneity based on assays of (i) inhibition of the replication of plasmids (and other supercoiled DNA) lacking oriC and (ii) conferral of dnaA protein dependence on the replication of an oriC plasmid. This specificity protein is indistinguishable from authentic E. coli topoisomerase I by several criteria: (i) molecular weight under denaturing conditions, (ii) relaxation activity on negatively supercoiled DNA, (iii) cleavage pattern of single-stranded DNA, (iv) specificity factor activity, and (v) neutralization of activity by antibody against topoisomerase I. One possible mechanism of the specificity action of topoisomerase I is destabilization of primers for replication except when they are preserved at an oriC sequence bound by dnaA protein and other replication proteins.     

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.     

The dnaK gene of Escherichia coli functions in initiation of chromosome replication.

A newly isolated dnaK mutant of Escherichia coli, which contains the mutation dnaK111, has been found to be conditionally defective in initiation of DNA replication. Mutant cells that were transferred to high temperature exhibited residual DNA synthesis before the synthesis stopped completely. Analysis of the DNA synthesized at high temperature by hybridization with probe DNAs for detection of DNA replicated in the origin (oriC) and terminal (terC) regions has revealed that this mutant is unable to initiate a new round of DNA replication at high temperature after termination of the round in progress. The cells exposed to high temperature were subsequently capable of initiating DNA replication at low temperature in a synchronous manner. DNA synthesis of this mutant became temperature resistant upon inactivation of the rnh gene, similar to that of dnaA mutants, although cell growth of the dnaK mutant with the inactive rnh gene remained temperature sensitive. The dnaK mutation prevented DNA synthesis of lambda bacteriophage at high temperature even in the absence of the rnh gene function.     

Homologous recombination and chromosomal rearrangements in Escherichia coli strains carrying a heterozygous tandem duplication]

Heterozygous tandem duplications formed in conjugational matings in Escherichia coli provides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deo operon of E. coli carrying deoA deoB::Tn5/deoC deoD thr::Tn9 alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3 mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ (or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of "adaptive" recombination.     

Different effects of mioC transcription on initiation of chromosomal and minichromosomal replication in Escherichia coli.

The mioC gene, which neighbors the chromosomal origin of replication (oriC) in Escherichia coli, has in a number of studies been implicated in the control of oriC initiation on minichromosomes. The present work reports on the construction of cells carrying different mioC mutations on the chromosome itself. Flow cytometry was employed to study the DNA replication control and growth pattern of the resulting mioC mutants. All parameters measured (growth rate, cell size, DNA/cell, number of origins per cell, timing of initiation) were the same for the wild type and all the mioC mutant cells under steady state growth and after different shifts in growth medium and after induction of the stringent response. It may be concluded that the dramatic effects of mioC mutations reported for minichromosomes are not observed for chromosomal replication and that the mioC gene and gene product is of little importance for the control of initiation. The data demonstrate that a minichromosome is not necessarily a valid model for chromosomal replication.