Evidence for a fixed termination site of chromosome replication in Escherichia coli K12.

We have investigated the possibility of a fixed terminus for bidirectional replication in Escherichia coli by determining whether a displacement of the chromosome replication origin results in an inversion of the direction of replication for markers located in the region where termination normally occurs.Three prophages have been used to mark four chromosomal sites: Mu-1, integrated in either malA (74 min) or malB (90 min); P2 in location H (43 min) and φ80 (27 min). Integrative suppression, promoted by a resistance transfer factor, resulted in origin displacements greater than 20 minutes in each direction. In the parental strains and in their integratively suppressed derivatives we have established, for each prophage: (a) the direction of replication (by hybridizing labelled Okazaki fragments to separated phage strands); (b) the relative frequency, in the exponential phase of growth (by DNA-DNA hybridization of long-term labelled DNA to denatured phage DNA).The following conclusions have been reached. (1) In conditions of integrative suppression, chromosome replication is bidirectional, starting from the inserted episome. (2) The direction of replication of each of the two prophages, P2 and φ80, is invariant in the termination region. (3) Marker frequency analysis has revealed that P2 prophage and φ80 prophage are on two different replication units.These results suggest that replication forks, travelling in either direction, must stop at a site located between 27 and 43 minutes on the genetic map, presumably the terminus of replication (tre).     

The Escherichia coli terB sequence affects maintenance of a plasmid with the M13 phage replication origin.

Replication initiated at the bacteriophage M13 origin can be affected by interaction of a properly oriented termination signal terB and the Tus protein. The effect can be alleviated by overproduction of the M13 replication gene protein II.     

Inititation and termination of chromosome replication in Escherichia coli subjected to amino acid starvation.

Initiation and termination of chromosome replication in an Escherichia coli auxotroph subjected to amino acid starvation were examined by following the incorporation of [3H]thymidine into the EcoRI restriction fragments of the chromosome. The pattern of incorporation observed upon restoration of the amino acid showed that starvation blocks the process of initiation prior to deoxyribonucleic acid synthesis within any significant portion of the EcoRI fragment which contains the origin of replication, oriC. In this experiment, no incorporation of [3H]thymidine into EcoRI fragments from the terminus of replication was observed, nor was it found when a dnaC initiation mutant was used to prevent incorporation at the origin which might have obscured labeling of terminus fragments. Thus amino acid starvation does not appear to block replication forks shortly before termination of replication. Attempted synchronization of replication initiation by including a period of thymine starvation subsequent to the amino acid starvation led to simultaneous incorporation of [3H]-thymidine into all EcoRI fragments within the 240-kilobase region that surrounds oriC. It is shown that the thymine starvation step allowed initiation and a variable, but limited, amount of replication to occur.     

A gene coding for a periplasmic protein is located near the locus for termination of chromosome replication in Escherichia coli

Hybrid plasmids carrying trg, the genetic locus in closest proximity to terC, coded for several polypeptides in addition to the Trg protein. Polypeptides of 59,000 and 61,000 apparent molecular weight were the most prominent products synthesized in minicells containing the hybrid plasmids. Analysis of the effects of deletions generated by a restriction endonuclease identified a region of DNA immediately adjacent to trg as the putative gene coding for the two polypeptides. Studies with whole cells and minicells showed that the 59,000-dalton polypeptide is a periplasmic protein. Analysis by limited proteolysis indicated that the two polypeptides are related, and a number of observations support the notion that the 61,000-dalton protein is a precursor form of the 59,000-dalton mature exported protein. The identification and characterization of a protein, in addition to Trg, which is produced by a gene in close proximity to terC emphasizes the fact that the region does contain intact and active genes.     

Large deletions in the region of the replication termination of Escherichia coli K-12 chromosome

The Escherichia coli strain PLK1427 (Henson, Kopp, Kuempel, 1984) was used in this work. It carries a deletion of 60 thousand pairs of nucleotides in the chromosomal region 30-31 min and a partially deleted prophage lambda rev cI875Sam7 integrated into the 30 min region, instead of the rac prophage. Among the mutants of PLK1427 strain selected for resistance to 42 degrees C, deletions extending about 4 min and affecting the loci nirR (29.3 min), zdc235::Tn10 (32.3 min) and zdd230::Tn9 (33.3 min) were found. Although the deletion mutants obtained affect the region of replication termination (terC) of the chromosome, they have no alterations in the growth rate. It was demonstrated that some deletions may be transferred and are capable of recombination, giving the wild type in transductional experiments with the mutant phage T4.     

On the termination of the DNA replication cycle in Escherichia coli.

The initiation of the DNA replication cycle in Escherichia coli requires protein synthesis. Marunouchi &; Messer (1973) have hypothesized that an additional protein synthesis step is required for the replication of the terminal segment of the chromosome, and that replication of this segment is a prerequisite for subsequent cell division. We have not confirmed the existence of a unique terminal segment using a protocol designed to label the hypothesized segment with [³H]dThd². Our protocol avoids the increased incorporation of [³H]dThd into DNA caused by abrupt increases in temperature, a complication implicit in the technique of Marunouchi &; Messer (1973).Treatment with nalidixic acid (an inhibitor of semiconservative DNA synthesis) in sufficient concentration to prevent replication of the postulated terminal segment prevents cell division but also causes loss of viability. This makes it difficult to correlate the effect of nalidixic acid on cell division with DNA synthesis inhibition alone.     

Control of cyclic chromosome replication in Escherichia coli.

The biochemical basis for cyclic initiation of bacterial chromosome replication is reviewed to define the processes involved and to focus on the putative oscillator mechanism which generates the replication clock. The properties required for a functional oscillator are defined, and their implications are discussed. We show that positive control models, but not negative ones, can explain cyclic initiation. In particular, the widely accepted idea that DnaA protein controls the timing of initiation is examined in detail. Our analysis indicates that DnaA protein is not involved in the oscillator mechanism. We conclude that the generations of a single leading to cyclic initiation is separate from the initiation process itself and propose a heuristic model to focus attention on possible oscillator mechanisms.     

The X chromosome is a hot spot for sexually antagonistic fitness variation

Sexually antagonistic alleles are selected discordantly between the sexes. Experimental evidence indicates that sexually antagonistic fitness variation is abundant in the genome of Drosophila melanogaster. Theory predicts that the X chromosome will be enriched with this type of variation. To test this prediction in D. melanogaster, we sampled, and cytogenetically cloned, 20 X chromosomes and compared their fitness variation to genome-wide levels. At the juvenile stage, in which gender roles are most similar, the X chromosome made no detectable contribution to genome-wide fitness variation. At the adult stage, in which gender roles diverge, the X chromosome was estimated to harbour 45% of the genome-wide fitness variation and 97% of the genome-wide sexually antagonistic variation. This genomic structure has important implications for the process of sexual selection because X-linked sexually antagonistic variation contributes to negative intersexual heritability for fitness, i.e. high-fitness males (females) produce, on average, low-fitness daughters (sons).     

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.     

The replication fork trap and termination of chromosome replication

Bacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a ‘replication fork trap’ to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over‐replication, and the optimization of post‐replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.     

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.     

The terminus of the Escherichia coli chromosome is flanked by several polar polar replication pause sites

Replication of two small ‘constrained’ regions of the Escherichia coli chromosome, one bordered by replication terminator T1 and the other by T2, displays normal velocity in the normal direction whereas it is much slower in the opposite direction (de Massy et al., 1987). The presence of multiple polar terminators has been investigated, using a bacteriophage λ derivative which provides a replication origin movable to predetermined loci and inducible on demand. The amount of DNA made from this induced origin was determined by in vivo labelling and hybridization to probes of the surrounding region. A redundancy of terminator-like sequences, or pause sites, has been disclosed. So far, two polar pause sites, in the same orientation and separated by 50 or 80 kb, have been localized on each side of the terminus region. The results are discussed in relation to previously observations indicating that these regions are refractory to genomic inversions.     

Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication

The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Involvement of Fis protein in replication of the Escherichia coli chromosome.

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.     

Growth rate-dependent control of chromosome replication initiation in Escherichia coli.

The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria. In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h). A low-thymine-requiring (thyA deoB) derivative of E. coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations. It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication. The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.     

Coordinate initiation of chromosome and minichromosome replication in Escherichia coli.

Escherichia coli minichromosomes harboring as little as 327 base pairs of DNA from the chromosomal origin of replication (oriC) were found to replicate in a discrete burst during the division cycle of cells growing with generation times between 25 and 60 min at 37 degrees C. The mean cell age at minichromosome replication coincided with the mean age at initiation of chromosome replication at all growth rates, and furthermore, the age distributions of the two events were indistinguishable. It is concluded that initiation of replication from oriC is controlled in the same manner on minichromosomes and chromosomes over the entire range of growth rates and that the timing mechanism acts within the minimal oriC nucleotide sequence required for replication.