Termination Of Chromosome Replication Research Articles

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.

Replication fork arrest and termination of chromosome replication in Bacillus subtilis.

Sporulation in Bacillus subtilis provided the first evidence for the presence of sequence-specific replication fork arrest (Ter) sites in the terminus region of the bacterial chromosome. These sites, when complexed with the replication terminator protein (RTP), block movement of a replication fork in a polar manner. The Ter sites are organized into two opposed groups which force the approaching forks to meet and fuse within a restricted terminus region. While the precise advantage provided to the cell through the presence of the so-called replication fork trap is not patently obvious, the same situation appears to have evolved independently in Escherichia coli. The molecular mechanism by which the RTP-Ter complex of B. subtilis (or the analogous but apparently unrelated complex in E. coli) functions is currently unresolved and subject to intense investigation. Replication fork arrest in B. subtilis, requiring RTP, also occurs under conditions of the stringent response at so-called STer sites that lie close to and on both sides of oriC. These sites are yet to be identified and characterized. How they are induced to function under stringent conditions is of considerable interest, and could provide vital clues about the mechanism of fork arrest by RTP-terminator complexes in general.

Bacterial chromosome segregation.

Recent years have witnessed a resurgence of interest in how the bacterial chromosome is organized and how newly replicated chromosomes are faithfully segregated into daughter cells on cell division. In the past, the problem with studying bacterial chromosomes was their lack of any obvious morphology, combined with the lack of ability to readily separate DNA replication and segregation functions into distinct stages like those observed in eukaryotic cells. This was due to the overlapping nature of these events in most bacterial systems used in the laboratory. The situation has now changed as new tools have become available that enable chromosomes and specific chromosomal sites to be labelled and monitored throughout the cell cycle, and this has led to rapid progress and the discovery of many unexpected results. Historically, chromosome segregation was thought to be achieved through passive processes where chromosomes were separated through some kind of membrane/cell wall attachment and were moved apart as the cell grew (Jacob et al., 1963). We now know that this is not the case and that there are specific mechanisms to actively partition chromosomes. This review will focus principally on the Gram-positive sporulating bacterium Bacillus subtilis, but will also cover work carried out on Escherichia coli, in which valuable information has been obtained, and will cover the events that occur on termination of chromosome replication, chromosome decatenation and chromosome separation.

Nucleoid structure and distribution in thermophilic Archaea.

Nucleoid structure and distribution in thermophilic organisms from the Archaea domain were studied. Combined phase-contrast and fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained Sulfolobus acidocaldarius and Sulfolobus solfataricus cells revealed that the nucleoids were highly structured. Different nucleoid distribution within the cells, representing different partition stages, was observed. The conformation of the nucleoids differed between exponentially growing and stationary-phase cells. Also, the stationary-phase cells contained two chromosomes, and the nucleoids occupied a larger part of the interior of the cells than in the exponentially growing cells. The part of the cell cycle during which fully separated nucleoids could be detected was short. Since the postreplication period is long in these organisms, there was a considerable time interval between termination of chromosome replication and completion of nucleoid separation, similar to the G2 phase in eukaryotic cells. The length of the visible cell constriction period was found to be in the same range as that of eubacteria. Finally, cell-cell connections were observed under certain conditions. Possible eubacterial, eukaryotic, and unique features of nucleoid processing and cell division in thermophilic archaea are discussed.

Cell cycle characteristics of thermophilic archaea.

We have performed a cell cycle analysis of organisms from the Archaea domain. Exponentially growing cells of the thermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius were analyzed by flow cytometry, and several unusual cell cycle characteristics were found. The cells initiated chromosome replication shortly after cell division such that the proportion of cells with a single chromosome equivalent was low in the population. The postreplication period was found to be long; i.e., there was a considerable time interval from termination of chromosome replication until cell division. A further unusual feature was that cells in stationary phase contained two genome equivalents, showing that they entered the resting stage during the postreplication period. Also, a reduction in cellular light scatter was observed during entry into stationary phase, which appeared to reflect changes not only in cell size but also in morphology and/or composition. Finally, the in vivo organization of the chromosome DNA appeared to be different from that of eubacteria, as revealed by variation in the relative binding efficiency of different DNA stains.

Definition and polarity of action of DNA replication terminators in Bacillus subtilis

The first stage in termination of chromosome replication in Bacillus subtilis involves arrest of the clockwise fork at the inverted repeat region (IRR), comprising the opposed IRI and IRII sequences, adjacent to the upstream region of the rtp gene, which encodes the replication terminator protein RTP. RTP binds to IRI and IRII. The ability of the IRR and its components to function as terminators, in conjunction with RTP, and their polarity of action have now been tested by the use of plasmids replicating in B. subtilis as unidirectional theta structures and into which potential terminator sequences were inserted in alternate orientations relative to fork movement. When the complete IRR was inserted into such plasmids and the new plasmids transferred into a B. subtilis strain overproducing RTP, it was able to block movement of a replication fork approaching from either direction. IRI and IRII were shown to function as polar terminators, each blocking movement of a fork when it approached from one particular direction but not the other. Furthermore, the polarity of action was in accordance with the IRR being able to operate as a replication fork trap. Thus, a fork approaching the IRR would pass through the first terminator encountered (IRI or IRII) and be halted by the second. The previously observed non-functioning of a particular orientation of chromosomal IRR as a fork arrest site probably reflects a limiting level of RTP in the cell. Interestingly, a 21 base-pair core sequence spanning a single RTP binding site within IRI (the 47 base-pair IRI contains 2 binding sites) was unable to arrest a fork approaching from either direction in the plasmid system. This suggests that both binding sites within an IR must be filled in order to function as an arrest site. It is possible that co-operative interaction between adjacent dimers within IRI or IRII provides the necessary conformation for causing fork arrest.

Probing the activation of the replicative origin of broad host-range plasmid R1162 with Tus, theE.coilanti-helicase protein

The E.coli Tus protein is an anti-helicase involved in the termination of chromosome replication. The binding site for this protein, ter, was cloned into derivatives of the broad host-range plasmid R1162. The ter site caused the orientation-specific termination of plasmid replication fork movement in cell extracts containing Tus. Plasmids were constructed so that two sites for initiation of R1162 replication flanked the iteron-containing domain of the origin. In these plasmids, the site next to the AT-rich region within the iteron-containing domain was more active. In addition, when ter was placed between the more active site and the iterons, initiation of replication from this site was specifically inhibited. The data support a model for entry of the essential, plasmid-encoded helicase at one side of the direct repeats, and for its movement primarily in one direction away from these repeats to activate the initiation sites for DNA replication.

Termination of chromosome replication in Bacillus subtilis

Termination of chromosome replication in Bacillus subtilis

A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site

The small basic protein encoded by the open reading frame adjacent to the terC site in the Bacillus subtilis chromosome and previously implicated in termination of the replication process was purified. Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.

An immobilized fork as a termination of replication intermediate in Bacillus subtilis

The structure of a DNA intermediate associated with termination of chromosome replication in Bacillus subtilis and derived from a unique BamHI 24.8 × 10 3 base-pair (bp) region of the chromosome has been investigated. The intermediate has properties expected for a forked structure. Gel electrophoresis followed by Southern transfer and hybridization to cloned DNA has shown it to comprise single strands of 15.4 × 10 3 bp and 24.8 × 10 3 bp, in approximately equimolar amounts. After purification away from the bulk of chromosomal DNA, electron microscopy of the intermediate established that 15% of the DNA was present as branched molecules and a significant proportion (11 of 31) of these contained two arms of matching length. The average dimensions (best estimates) of this unique class of Y-shaped molecule were 9.5(± 0.3) × 10 3, 15.1(± 0.4) × 10 3 and 24.6(±0.6) × 10 3 bp for the stem, arms and end-to-end length, respectively. These values are consistent with the single strand composition of the intermediate as found. Furthermore, hybridization of the single strands to DNA from known locations within the BamHI 24.8 × 10 3 bp region has established the orientation of the forked intermediate relative to the genetic map. The intermediate presumably reflects the immobilization of the clockwise replication fork within the 24.8 × 10 3 bp region, at a location approximately 15.4 × 10 3 bp from the right end.

A unique DNA intermediate associated with termination of chromosome replication in Bacillus subtilis

A unique DNA structural intermediate associated with termination of replication in Bacillus subtilis has been identified. This was achieved by probing for the Bam HI DNA fragment within which the terminus of the chromosome ( terC) resides. The intermediate migrates more slowly than the bulk of digested chromosomal DNA upon electrophoresis. It is more readily detected in DNA from a merodiploid strain in which the clockwise fork should be immobilized at terC for longer than usual, and it is destroyed by S1 nuclease. The intermediate may be a forked or related structure. These findings provide evidence for the existence of a specific, sequence-based termination site that blocks or severely impedes the movement of at least the clockwise fork, which is the first to encounter it.

Initiation of wall assembly sites in Streptococcus faecium.

In electron micrographs of replicas of Streptococcus faecium, sites of wall growth are located between pairs of raised equatorial bands. Analysis of cells taken from cultures with mass doubling times between 30 and 125 min indicates that rounds of wall synthesis are initiated at a time close to division, which is temporally unrelated to the initiation or termination of chromosome replication. Growth sites are initiated at a relatively constant volume independent of growth rate when the volume contained within the two segments of wall adjoining an equatorial band marker approaches ca. 0.26 micrometer 3.

The cell cycle in Escherichia coli B/r

Cell division and DNA synthesis were measured in synchronous cultures of E. coll B/r growing in glucose minimal medium at 37 °. The kinetic curves were analysed in order to find the variability of replication initiation, termination, and cell division events during the cell cycle. It is inferred that under the conditions used, cells begin to divide 17 min ( D 0 = minimum D-period) after each termination of chromosome replication with a constant probability per unit of time (half-life = 4·5–6 min). This randomness produces an asymmetric frequency distribution of D-periods, similar but mirror-symmetric frequency distributions of initiation and termination periods, a symmetric, non-Gaussian distribution of interdivision intervals, and complex kinetic changes in the rate of DNA synthesis as a function of cell age. The results suggest that replication and division are precisely controlled with respect to mass accumulation, and the apparent variability of cell cycle events would only result from the use of the time of cell separation as a reference point for the definition of cell age rather than initiation or termination of replication.

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.

Division septation in the absence of chromosome termination in Bacillus subtilis

Germinating spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis, TsB134 and dna-1(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in Cs 2SO 4 gradients. The replication state of origin ( purA), intermediate ( leuA) and terminus ( metB) markers at the times of 5-bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl. The DNA replication inhibitor, 6-( p-hydroxyphenylazo)-uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 μ m) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrations of HPUra, 40 and 100 μ m, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson & Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.

Morphological analysis of the division cycle of two Escherichia coli substrains during slow growth

Morphological parameters of the cell division cycle have been examined in Escherichia coli B/r A and K. Whereas the shape factor (length of newborn cell/width) of the two strains was the same at rapid growth (doubling time, tau, less than 60 min), with decreasing growth rate the dimensions of the two strains did change so that B/r A cells became more rounded and B/r K cells became more elongated. The process of visible cell constriction (T period) lasted longer in B/r A than in B/r K during slow growth, reaching at tau = 200 min values of 40 and 17 min, respectively. The time between termination of chromosome replication and cell division (D period) was found to be longer in B/r A than in B/r K. As a result, in either strain completion of chromosome replication seemed always to occur before initiation of cell constriction. Nucleoplasmic separation did not coincide with termination as during rapid growth but occurred in both strains within the T period, about 10 min before cell division.

Initiation and termination of chromosome replication at 45 degree C in a temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134

Deoxyribose nucleic acid transfer experiments showed that upon shifting Bacillus subtilis TsB134 to 45 degree C, initiation of new rounds of replication was effectively blocked and the majority of existing rounds terminated.

Estimation of the D period from residual division after exposure of exponential phase bacteria to chloramphenicol.

Values of the D period, between termination of chromosome replication and cell division, were determined from measurements of residual cell division after exposure of exponential phase cultures of Escherichia coli B/r and K12 and of Salmonella typhimurium to chloramphenicol. The results obtained by this method were compared with earlier results for E. coli B/r obtained from measurements of DNA content per cell and were found to be almost identical. For each, values of the D period were independent of growth rate, and the average value of D-26.1 plus or minus 1.2 min obtained by residual division is in good agreement with the value of 25 min obtained earlier. These results indicate that the method of residual division provides a good measure of the duration of the D period. Values of D were also independent of growth rate for each of the other strains.

Coupling between chromosome completion and cell division in Escherichia coli.

The relationship between termination of chromosome replication and cell division was investigated in Escherichia coli B/r. Synchronous cultures of E. coli B/r growing in glucose minimal medium or subjected to a nutritional shift-up were exposed to chloramphenicol, rifampin, mitomycin C, or nalidixic acid, and the ability of cells to divide in the presence of the inhibitors was measured. It was found that cell division became resistant to inhibitors of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis at approximately the same stage in the division cycle in all situations investigated. When the synchronous glucose-grown cultures were temporarily exposed to chloramphenicol early in the division cycle and then exposed to mitomycin C or nalidixic acid immediately after removal of chloramphenicol, the cells did not divide. In contrast, when DNA synthesis was inhibited by thymine starvation immediately after temporary exposure to chloramphenicol, cells divided. The results suggest that DNA chain elongation is completed in some cells in the absence of protein synthesis, but that additional steps involving specific RNA or protein synthesis, or both, may be required for processing the chromosomal structures to the form which is necessary for division. This processing, which normally occurs concurrent with DNA synthesis and is prevented by inhibitors of DNA synthesis, may trigger division. Alternatively, in the absence of protein synthesis, all aspects of chromosome formation may be completed, but final transcriptional events which are essential for division cannot take place until the complete synthesis of a critical amount of specific proteins.

Bidirectional termination of chromosome replication in Escherichia coli.

Autoradiography was used to study the termination of replication of the circular chromosome of Escherichia coli. The experiments were conducted with cells in which termination occurred with a moderate amount of synchrony. Grain tracks were observed that demonstrated the approach at the replication terminus of the two replication forks involved in bidirectional replication. Other grain tracks were formed by replication forks that had met at the replication terminus. The frequency at which these patterns were observed indicates that most, if not all, terminations occur with both replication forks reaching the terminus at approximately the same time.