Coordination between chromosome replication and cell division in Escherichia coli.

Cell division properties of Escherichia coli B/r containing either a dnaC or a dnaI mutation were examined. Incubation at nonpermissive temperature resulted in the eventual production of cells of approximately normal size, or slightly smaller, which lacked chromosomal DNA. The cell division patterns in cultures which were grown at permissive temperature and then shifted to nonpermissive temperature were consistent with: first, division and equipartition of chromosomes by cells which were in the C and D periods at the time of the shift; second, an apparent delay in cell division; and third, commencement of the formation of chromosomeless cells. In glucose-grown cultures of the dnaI mutant, production of chromosomeless cells continued for at least 120 min, whereas in the dnaC mutant chromosomeless cells were formed during a single interval between 110 and 130 min after the temperature shift. The results are discussed in light of the hypothesis that replication of a specific chromosomal region is not an obligatory requirement for the initiation and completion of the processes leading to division in a cell which contains at least one functioning chromosome.     

Connecting chromosome replication with cell growth in bacteria

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Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.     

Relation of DNA replication and cell division frequency in a population of microorganisms to the cell growth rate

DNA replication and the frequency of cell division were studied in a microbial population in relation to the rate of cell growth. The relationship is based on the law of cell biomass linear increase during the cell cycle and on the exponential law of mean cell mass increase, and depends on the specific rate of population growth. The cell mass in the initiation of DNA replication is correlated with the number of initiation points basing on the Cooper-Helmstetter theory of DNA replication and taking account of the linear growth of mass in one cell. Possible applications of these relationships are discussed.     

A role for the octameric ring protein,Translin, in mitotic cell division

The octameric ring protein, Translin, demonstrates marked similarities to the family of helicase enzymes regarding its quaternary organization and dimerization of subunits. Here we show that the level of Translin closely parallels the proliferative state in various cell types. Expression is periodic during the cell cycle, with protein synthesis becoming maximal in the S and mitosis phases, consistent with a role in cell division. Moreover, induced overexpression of Translin was found to accelerate cell proliferation. Confocal microscopic analysis revealed that Translin is localized at the centrosomes at prophase and the mitotic spindle at metaphase, then translocating to the spindle midbodies during cytokinesis. This novel localization is attributable to specific interactions with microtubules of the mitotic spindles, and especially gamma-tubulin. The results suggest that Translin participates in processes ensuring the segregation of chromosomes and cytokinesis.     

A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli.

Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40 degrees C but completely repressed at 27 degrees C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTP-binding proteins as important regulators of cell growth and division may be ubiquitous in nature.     

A fixed amount of chromosome replication needed for premature division septation in Bacillus subtilis

Germinating spores of the temperature-sensitive DNA initiation mutant of Bacillus subtilis, TsB134, were allowed to undergo a single round of replication, at a high and low level of thymine, by shifting to the non-permissive temperature shortly after its initiation. The rate of replication at the low thymine level was approximately half that at the other, but there was no significant difference in the rate of cell mass increase. The round of replication in each case was blocked at various stages by 6-(p-hydroxyphenylazo)uracil and outgrown cells examined at a later time for the frequency of central division septation. It was found that the same average amount of replication (fraction of the round) was required in both cases for premature division septation to proceed.     

The role for zinc in replication protein A

Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. The proteolytic core of this domain comprised amino acids 432-616. X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (amino acids 43-171), and RPA14 had strong DNA binding activity. When properly coordinated with zinc, the trimer's affinity to ssDNA was only 3-10-fold less than that of the ssDNA-binding domain in the middle of RPA70. However, the DNA-binding activity of the trimer was dramatically reduced in the presence of chelating agents. Our data indicate that (i) Zn(II) is essential to stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses DNA-binding activity, which is modulated by Zn(II); and (iii) ssDNA binding by the trimer is a synergistic effect generated by the RPA70-CTD and RPA32.     

A role of topoisomerase II in linking DNA replication to chromosome condensation

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis-dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II-containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter

Bacterial chromosome partitioning and cell division are tightly connected cellular processes. We show here that the Caulobacter crescentus FtsK protein localizes to the division plane, where it mediates multiple functions involved in chromosome segregation and cytokinesis. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. These results suggest that the interdependence between chromosome partitioning and cell division in Caulobacter is mediated, in part, by the FtsK protein.     

A role for DNA primase in coupling DNA replication to DNA damage response.

The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.     

Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation

Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP‐on‐Chip and bioinformatics, we have identified a consensus Noc‐binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc–YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc–YFP and conferred a severe Noc‐dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.