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.     

Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis

Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.     

Internal reinitiation of translation in polar mutants of the trpB gene of Salmonella typhimurium.

Summary The trpB gene of S. typhimurium codes for the bifunctional component II subunit of the AS-PRT complex which catalyzes the first two steps of tryptophan biosynthesis. It has previously been shown that the amino-terminal 40% of the component II molecule possesses the catalytic sites determining glutamine amidotransferase (GAT) activity, demonstrable indirectly by complementation with component I, the product of trpA, in the synthesis of anthranilic acid from chorismic acid and glutamine (AS activity), while its carboxy-terminal 60% possesses the catalytic sites determining anthranilate-PRPP phosphoribosyl transferase (PRT) activity, demonstrable by direct enzymatic assay. Here we further demonstrate the functional independence of the two regions of the component II subunit by providing evidence for the existence of monofunctional (GAT^-, PRT⁺) carboxy-terminal restart fragments of component II in certain chain terminating trpB mutants. Nonsense and frameshift mutants of the operator-proximal portion (region 1) of trpB have been found to grow well in media supplemented with anthranilic acid, implying the presence of PRT activity in the cell. Analysis of extracts of these strains has demonstrated the presence of low, but variable levels of PRT activity, but no GAT activity. Correlation of the map location of these mutations with the intensity of their polar effects on the expression of operator-distal genes suggests the existence of at least two gradients or units of polarity within region 1. Furthermore, in double mutant polarity tests, multiplicatiove polar effects were found in certain region 1 trpB-trpB double mutants strains. Taken together, these results lead us to conclude that at least two sites for reinitiation of translation exist within region 1 of trpB which can be activated by the presence of a nearby chain terminating codon. Such reinitation leads to the synthesis of labile carboxy-terminal restart fragments of component II which possess PRT function, but lack GAT function.     

Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.     

Mechanisms of polar arrest of a replication fork

A DNA replication terminator sequence blocks an approaching replication fork when the moving replisome approaches from just one direction. The mechanism underlying polar arrest has been debated for years, but recent work has helped to reveal how a replication fork is blocked in Escherichia coli . Early work suggested that asymmetric interaction between terminator protein and terminator DNA contributes to polar fork arrest. A later study demonstrated that if the terminator DNA is partially unwound, the resulting melted DNA could bind tightly to the terminator protein, suggesting a mechanism for polar arrest that involves a locked complex. However, recent evidence suggests that the terminator protein–DNA contacts are not sufficient for polar arrest in vivo . Furthermore, polar arrest of a replication fork still occurs in the absence of a locked complex between the terminator protein and DNA. In E. coli and Bacillus subtilis , the bound terminator protein makes protein–protein contacts with the replication fork helicase, and these contacts are critical in blocking progression of the advancing fork. Thus, we propose that interactions between the replication fork helicase and terminator protein are the primary mechanism for polar fork arrest in bacteria, and that this primary mechanism is modulated by asymmetric contacts between the terminator protein and its cognate DNA sequence. In yeast, terminator sequences are present in rDNA non-transcribed spacers and a region immediately preceding the mating type switch locus Mat1, and the mechanism of polar arrest at these regions is beginning to be elucidated.     

Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences

Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions. Since these regions also harbour polar replication terminator-like sequences or pause sites, we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators. We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.     

Base Composition Skews, Replication Orientation, and Gene Orientation in 12 Prokaryote Genomes

Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes. Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication. The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species. In M. genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome. Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication. Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated. Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3. Received: 2 February 1998 / Accepted: 15 June 1998     

Termination Structures in the Escherichia coli Chromosome Replication Fork Trap

The Escherichia coli chromosome contains two opposed sets of unidirectional DNA replication pause (Ter) sites that, according to the replication fork trap theory, control the termination of chromosome replication by restricting replication fork fusion to the terminus region. In contrast, a recent hypothesis suggested that termination occurs at the dif locus instead. Using two-dimensional agarose gel electrophoresis, we examined DNA replication intermediates at the Ter sites and at dif in wild-type cells. Two definitive signatures of site-specific termination—specific replication fork arrest and converging replication forks—were clearly detected at Ter sites, but not at dif. We also detected a significant pause during the latter stages of replication fork convergence at Ter sites. Quantification of fork pausing at the Ter sites in both their native chromosomal context and the plasmid context further supported the fork trap model.     

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).     

Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites

In bacteria, Ter sites bound to Tus/Rtp proteins halt replication forks moving only in one direction, providing a convenient mechanism to terminate them once the chromosome had been replicated. Considering the importance of replication termination and its position as a checkpoint in cell division, the accumulated knowledge on these systems has not dispelled fundamental questions regarding its role in cell biology: why are there so many copies of Ter, why are they distributed over such a large portion of the chromosome, why is the tus gene not conserved among bacteria, and why do tus mutants lack measurable phenotypes? Here we examine bacterial genomes using bioinformatics techniques to identify the region(s) where DNA polymerase III‐mediated replication has historically been terminated. We find that in both Escherichia coli and Bacillus subtilis, changes in mutational bias patterns indicate that replication termination most likely occurs at or near the dif site. More importantly, there is no evidence from mutational bias signatures that replication forks originating at oriC have terminated at Ter sites. We propose that Ter sites participate in halting replication forks originating from DNA repair events, and not those originating at the chromosomal origin of replication.     

Replication of ColE2 and ColE3 plasmids: The regions sufficient for autonomous replication

Summary We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.     

DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex

Plasmid pAL5000 represents a family of relatively newly discovered cryptic plasmids in gram-positive Actinomycetes bacteria. The replication regions of these plasmids comprise a bicistronic operon, repA-repB, encoding two replication proteins. Located upstream is a cis-acting element that functions as the origin of replication. It comprises an ~200-bp segment spanning two binding sites for the replication protein RepB, a low-affinity (L) site and a high-affinity (H) site separated by an ~40-bp spacer sequence. The trajectory of the DNA in the RepB-origin complex has been investigated, and it has been found that the origin undergoes significant bending movements upon RepB binding. RepB binding not only led to local bending effects but also caused a long-range polar curvature which affected the DNA sequences 3′ to the H site. These movements appear to be essential for the in-phase alignment of the L and H sites that leads to the formation of a looped structure. A novel property of RepB unearthed in this study is its ability to form multimers. This property may be an important factor that determines the overall trajectory of the DNA in the RepB-origin complex. The results presented in this study suggest that the origins of replication of pAL5000 and related plasmids are highly flexible and that multimeric, RepB-like initiator proteins bind the origin and induce local deformations and long-range curvatures which are probably necessary for the proper functioning of the origin.     

Genetic Organization of the Basic Replicon of Plasmid pMTH4 of a Facultatively Methylotrophic Bacterium <Emphasis Type="Italic">Paracoccus methylutens</Emphasis> DM12

Two functional regions within the basic replicon of plasmid pMTH4 of Paracoccus methylutens DM12 have been distinguished that are responsible for the replication of the plasmid (REP) and its stabilization (STA). In the REP region, a gene encoding the putative replication initiation protein RepA has been identified, with the highest similarity to the replication protein of plasmid pALC1 (Paracoccus alcaliphilus). The potential origin of replication (oriV), consisting of five long repeated sequences (iterons) as well as putative DnaA and IHF boxes, has been localized in the promoter region of the gene repA. The STA region was found to ensure stability for heterogeneous plasmid pABW3 that is unstable itself in paracocci. The mini-STA region (850 bp) contains two short open reading frames, one of which shows similarity to the RelB protein of Escherichia coli. Our investigations suggest that the stabilizing system of pMTH4 is based on the toxin and antidote principle.     

Morbillivirus ecology in polar bears (Ursus maritimus)

Polar bear (Ursus maritimus) morbillivirus infection was initially reported by Follmann and co-workers in 1996, based upon serologic results using canine distemper virus (CDV). The impetus for the evaluation of polar bear populations for morbillivirus infections was prompted by epidemics of canine distemper-like disease in seal populations in the north Atlantic regions of Greenland, Europe, and Russia. Since marine morbilliviruses have been further characterized into three major species, phocine distemper virus (PDV), dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV), it was of value to determine the origin of the polar bear infection. One hundred serum samples were selected from a group of sera collected from regions of Alaska and Russia and tested by differential serum neutralization assay against the three major marine morbilliviruses and CDV, to determine the predominant virus infecting the polar bear. Polar bears had higher serum antibody titers to CDV than they did to PDV, DMV, and PMV. These data suggest that polar bears are being infected with a morbillivirus of terrestrial origin. Furthermore, based on the high serum antibody prevalence in the population, the virus may be indigenous to the polar bear and not necessarily the result of interspecies transmission from other arctic mammals susceptible to CDV and/or marine morbilliviruses. Accepted: 20 December 1999     

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2

Summary A DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc ₂ incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc ₂ incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.     

Mapping replication units in animal cells

A general approach for assaying the in vivo direction of replication for any DNA segment has been developed. This technique allows the scanning of genomic regions to detect bidirectional tail-to-tail replication, indicating the presence of a functional origin. By this criterion we identified the approximate positions of two origin sites downstream of the Chinese hamster DHFR gene. Further mapping revealed areas of head-to-head replication, signifying locations of replication termination and thus defining the landmarks of a complete animal cell replicon. Genetic proof for the existence of the DHFR origin was obtained by showing that this region serves as a bidirectional DNA synthesis initiation point following its integration into other sites in the genome by transfection. To show the general applicability of this methodology, we studied the APRT domain. Replication mapping together with the use of deletion mutants allowed the identification of an origin at a far-upstream locus.     

Intrinsically bent DNA sites in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-β. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-β region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates

Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein). Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded. However, the other pause sites are in regions that are not obviously involved in secondary structure. The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides. At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions. The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing. In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing. This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.     

Two early replicated,developmentally controlled genes of Physarum display different patterns of DNA replication by two-dimensional agarose gel electrophoresis

The nature of replication origins in eukaryotic chromosomes has been examined in some detail only in yeast, Drosophila, and mammalian cells. We have used highly synchronous cultures of plasmodia of the myxomycete Physarum and two-dimensional agarose gel electrophoresis to examine replication of two developmentally controlled, early replicated genes over time in S-phase. A single, discrete origin of replication was found within 4.8 kb of the LAV1-5 gene, which encodes a homolog of profilin. In contrast, the LAV1-2 gene appears to be surrounded by several origins. Two origins were identified within a 15 kb chromosomal domain and appear to be inefficiently used. Replication forks collide at preferred sites within this domain. These terminating structures are long lived, persisting for at least 2 h of the 3 h S-phase. Analysis of restriction fragment length polymorphisms (RFLPs) within the LAV1-2 domain indicates that replication of alleles on different parental chromosomes is a highly coordinated process. Our studies of the these two early replicated, plasmodium-specific genes indicate that both a fixed, narrow origin region and a broader zone containing two closely spaced origins of DNA replication occur in Physarum.