Aparataje conceptual

In E. coli the two replication forks that begin at oriC and polymerize bidirectionally stop in a region of the chromosome opposite oriC, called Ter, the termination region (Fig. 3.14).

After termination, the sister chromosomes separate and partition (segregate) to opposite halves of the cell. The termination region con-tains specifi c sequences of nucleotides called Ter sequences.

The Ter sites are quite unusual because they allow replication to proceed in only one direc-tion! (Note that in Fig. 3.14 the arrows refer to the direction of replication, not the movement of the replication forks, which are station-ary within the replisome.) This property can account for termination at the Ter sites. For example, assume that there are two Ter sites, which are located next to each other, with per-haps a short segment of DNA between them.

Suppose Ter site 1 allows replication to proceed clockwise but not counterclockwise, and Ter site 2 allows counterclockwise replication but not clockwise replication. Thus, if clockwise replication arrives at site 2, it will stall; and if counterclockwise replication arrives at site 1, it will stall. Consequently, bidirectional replica-tion will meet at site 1 or site 2 or somewhere between them.

As shown in Fig. 3.14, the situation in E. coli is a little bit more complicated because it has six Ter sites, sites are divided into two groups of three: one group (TerA, TerD, and TerE) prevents counterclockwise replication, and the second group (TerC, TerB, and TerF), prevents clockwise replication. As explained in the legend to Fig. 3.14, the presence of three Ter sites per replication fork direction provides a backup in case the replication fork gets by one of these sites. A protein, which in E. coli is called the Tus (terminator utilization substance) protein binds to the Ter site and imposes the one-way travel. Tus does this by

Fig. 3.14 Termination of DNA replication in E. coli.

The arrows refer to the direction of replication, rather than to movement of the replication forks. A terminator region exists 180° opposite the origin. In the terminator region there are nucleotide sequences that allow replication in only one direction. Thus, the terminator sequences are said to be polar. E. coli has six terminator (Ter) sites. These are, in the sequence in which they exist in the DNA, TerE, TerD, TerA, TerC, TerB, and TerF. On one side of the terminator region are TerE, TerD, and TerA, and TerC, TerB, and TerF occupy the opposite side. The fi gure shows two replication forks. Data discussed earlier in this section support a model in which the replication forks are stationary within a DNA synthesizing “fac-tory” called the replisome. Unreplicated DNA enters the replisome and passes through the stationary rep-lication forks. TerC, TerB, and TerF inhibit clock-wise replication, and TerA, TerD, and TerE inhibit counterclockwise replication. Therefore, there are three chances to stop replication in the termination region. For example, if counterclockwise replica-tion moves past TerA, it will stop at TerD or TerE.

Consequently, replication from both directions will stop at one of the termination sites or a site between them. A protein, called the Tus protein in E. coli, binds to Ter and prevents the helicase from proceed-ing in one direction, thus accountproceed-ing for the polar-ity of inhibition of movement of the replication fork.

Notice the site marked dif. It is here that recombina-tion takes place to separate chromosomes that have undergone an unequal number of recombinations during replication.

inhibiting the replicative helicase (DnaB).^17–19 Both Ter sites and the Ter-binding protein have been well studied in B. subtilis. See note 20 for similarities and differences with respect to E. coli.

refl ecting the fact that because the sister chro-mosomes do not partition to opposite cell poles, they can both be in the same daughter cell upon cell division. Under these conditions, the nucle-oids are single nuclenucle-oids and are not larger than normal.

Chromosome separation

Chromosome separation requires the separa-tion of the two sister chromosome dimers, said to be catenated, by topoisomerase IV. The pro-cess is called decatenation. If there has been an unequal number of crossover events during DNA replication, then the sister chromosomes are covalently linked and must be separated by recombination by XerC and XerD.

Recombination occurs at dif sites. A key pro-tein here is the cell division propro-tein FtsK, which is localized to the septum and recruits other cell division proteins before cell division. FtsK ensures that XerCD and topoisomerase IV act on the DNA at midcell during the fi nal stages of cell division. A discussion of how this might occur can be found later (see subsection 3, which considers the catalytic activity on this enzyme).

1. Topoisomerase IV

When replication is complete (at about 180°

from the start site), the two completed DNA molecules are linked as two monomeric circles in a chain; that is, they are catenated and must be separated from each other (Fig. 3.15). The process is called decatenation. In E. coli and Salmonella typhimurium, the decatenation of two monomeric interlinked chromosomes requires the action of a DNA gyrase–like enzyme called topoisomerase IV.²¹ Topoisomerase IV mutants are temperature sensitive for growth;

that is, they grow at 30 °C but die at 42 °C.

At the restrictive temperature, cell division is inhibited and the mutants form elongated cells, often with unusually large nucleoids (revealed by DNA staining) in midcell or as several nucleoid masses unequally distributed in the elongated cells.²² The nucleoids are large because the two daughter nucleoids are not able to separate. The fi lamentous cells may divide in regions where there is no DNA to produce anucleate cells. Inhibition of cell division result-ing in elongated or fi lamentous cells is typical of mutants that have a block in DNA replica-tion or separareplica-tion of daughter chromosomes.

This refl ects the coupling between cell division 3.1.6 Chromosome separation and

partitioning; some general principles First, some defi nitions: chromosome separation refers to the detachment of the newly replicated chromosomes from one another, and chromo-some partitioning refers to the segregation of sister chromosomes to opposite halves of the cell prior to cell division.

Chromosome separation can take place in one of two ways. If the linkage between the daughter chromosomes is noncovalent, then topoisomerases such as topoisomerase IV sepa-rate the sister chromosomes, as we shall discuss shortly. If an unequal number of recombina-tions have occurred between the sister chro-mosomes, then the sister chromosomes are covalently linked and a site-specifi c recombina-tion must occur. (See later subsecrecombina-tion entitled 2.

Site-specifi c recombination at dif.)

Chromosome partitioning is not a well-un-derstood process in prokaryotes because a spin-dle apparatus similar to that which separates sister chromosomes in eukaryotes is not pres-ent. This section discusses current ideas about how separation and partitioning of daughter chromosomes occur in prokaryotes. Section 3.1.7 describes chromosome partitioning in B.

subtilis, and Section 3.1.8 describes chromo-some partitioning in C. crescentus.

Phenotypes of partition mutants

The genes involved in chromosome partition-ing have been discovered by analyzpartition-ing mutants.

Mutants defective in chromosome partitioning can be defective in any one of several genes: some are involved in detaching the chromosomes from each other, some are involved in the par-tition of the detached chromosomes, and some are even involved in the replication of DNA, because only fully replicated DNA molecules can detach from one another. Depending upon whether the defect is in detachment or partition of chromosomes, the phenotype differs. If the defect is in detaching the sister chromosomes from each other, then the phenotype includes elongated cells with an unusually large nucleoid mass positioned near the cell center. Anucleate cells may also form, as discussed next.

On the other hand, if the sister chromosomes can detach but cannot partition to opposite poles, the phenotype is an increased production of anucleate cells of the size of a newborn cell,

they chromosomes are covalently linked as a cir-cular dimer to each other and decatenation can-not separate them. Under these circumstances, the DNA molecules must be separated by site-specifi c recombination by using enzymes called recombinases. E. coli has a locus called dif (dele-tion-induced fi lamentation), which lies in the replication terminus (Ter) region.²⁵ The recom-binational event that separates the two sister chromosomes that have undergone an unequal number of recombinations during replication takes place in this Ter region (Fig. 3.16).

The site-specifi c recombination is catalyzed by two recombinase proteins, XerC and XerD, both of which are required.²⁶ The recombinases are activated by an E. coli cell division protein called FtsK, which is localized at midcell with other cell division proteins. (See Section 2.6.2 for a discussion of FtsK and the other cell divi-sion proteins in E. coli, and ref. 27 for a discus-sion of the multiple roles of FtsK in cell dividiscus-sion and chromosome segregation.) Homologues of XerC and XerD have been found in a wide variety of bacteria. Mutations in dif are viable, and most of the cells are normal. This is to be expected, since dif is required only when there has been an unequal number of recombinations.

However, approximately 10% of the cells are fi lamentous with unusually large nucleoids, indicative of a failure of the newly replicated chromosomes to separate. As expected, xerC and xerD mutants show the same phenotype as dif^– mutants.

3. Topoisomerase IV catalyzes decatenation primarily at dif in the presence of XerC, XerD, and FtsK

The activity of topoisomerase IV can take place at several chromosomal sites; but in the presence of FtsK, XerC, and XerD, decatenation takes place preferentially at midcell at the dif site.²⁸ However, unlike site-specifi c recombination, which requires XerC and XerD, decatenation at dif does not require the cell division protein FtsK, but is simply favored at dif because of FtsK. Why should topoisomerase IV preferentially cleave at dif? There have been two suggestions.²⁸ One suggestion is that the dif/XerC/XerD complex might increase the affi nity of topoisomerase IV for the dif site. A second suggestion is that topoi-somerase IV is part of a decatenation/resolution complex that forms at dif.

and DNA metabolism as described in note 23.

(Topoisomerase IV mutants have been isolated in C. crescentus, but the phenotype differs from that of E. coli and S. typhimurium mutants because checkpoints exist in the Caulobacter cell cycle.²⁴) Topoisomerase IV makes a double-stranded break in one of the DNA molecules, and the unbroken molecule is passed through the break (Fig. 3.15). This is followed by seal-ing the break (see section entitled Attachseal-ing the Okazaki fragments to each other).

2. Site-specifi c recombination at dif

If an unequal number of recombinations have occurred between sister circular chromosomes, Fig. 3.15 Separating catenated daughter circles after DNA replication by using type IV topoisomerase.

The reaction is catalyzed by type IV topoisomerase (similar to DNA gyrase). The enzyme catalyzes a double-stranded cut in one of the duplexes. Then the unbroken duplex passes through the gap. The gap is sealed, and the DNA molecules have become sepa-rated. Mutants defective in topoisomerase IV are temperature sensitive for growth and show abnor-mal nucleoid segration. The phenotype is called Par^–. See ref. 21.

Chromosome partitioning in eukaryotes It is worthwhile to summarize how chromosome partitioning takes place in eukaryotes before discussing how the process occurs in prokary-otes. In eukaryotes, chromosome replication is completed to form two sister chromatids bound along their length in the S phase of the cell cycle.

The sister chromatids remain together dur-ing the G₂ phase, which follows the S phase.

Following the G₂ phase is the M phase (mito-sis), and this is when the sister chromatids parti-tion. The M phase consists of nuclear division and cytokinesis. Partitioning occurs because the sister chromatids are attached to microtu-bule spindle fi bers that pull them apart. This occurs during the anaphase portion of mitosis.

Specifi cally, the sister chromatids are separated (and are now called chromosomes) and pulled to opposite cell poles by the shortening of the microtubular spindle fi bers in the mitotic spin-dle. Each chromatid is attached to the microtu-bule spindle fi bers via a protein complex called a kinetochore, which binds to a subset of micro-tubules in the mitotic spindle. The kinetochore is located at a highly condensed, constricted region called a centromere.

Chromosome partitioning in prokaryotes In prokaryotes, “chromosome partitioning”

refers to the segregation of sister chromosomes toward opposite cell poles in preparation for cell division. (Bacterial chromosomes are often referred to as nucleoids.) There is no evidence in bacteria for a device similar to the eukaryotic mitotic spindle to partition sister chromosomes.

So how do bacteria partition sister chromo-somes faithfully? This is a very active area of research, and the literature provides detailed information about the similarities and differ-ences with respect to what has been learned about B. subtilis, E. coli, and C. crescentus.^29–33 Certain generalizations can be made. First, it is Fig. 3.16 Site-specifi c recombination at

termina-tion to separate chromosomes that have undergone unequal numbers of recombinations. Simplifi ed drawing of recombination at dif to resolve a DNA dimer into monomers. As discussed in Section 3.1.5, the replication forks are stationary and remain close to each other in the replisome, a DNA-synthesizing

“factory.” The unreplicated DNA is fed through the forks. The arrows depict the direction of polymer-ization of DNA. (A) Unequal numbers of recom-binations have taken place covalently, linking the two circles to form a dimer. (B) Recombination is catalyzed by recombinases acting at the dif sites in the termination (Ter) regions. The chromosomes are then segregated to opposite cell poles. A dif⁻ mutant produces fi laments and anucleate cells as well as nor-mal cells. As the fi gure indicates, it is believed that the

recombination event at dif can proceed in both direc-tions. However, monomer formation is favored.

Perhaps the segregation of the monomers ensures that the newly replicated dif sites cannot recombine.

For recombination at dif to resolve the dimers, dif must be at its original site in the terminus region. If a copy of dif is reinserted in a different chromosomal site in a dif^– strain, the cells still show the dif^– phe-notype.

noted that partitioning (segregation) takes place concurrent with replication of the DNA.

The DNA is replicated in a stationary “fac-tory,” called the replisome, which in E. coli or B. subtilis is assembled at or near midcell when DNA replication is initiated and is thought to be anchored there. For a review of replisomes, see ref. 34. When DNA synthesis is fi nished, the replisome is disassembled. The unreplicated DNA threads through the replisome, and the newly duplicated portions move away from the anchored replication forks within the replisome and partition toward opposite poles of the cell.

Since the origins (ori sites) are replicated fi rst, they leave the replisome fi rst and very quickly are moved to opposite halves of the cell near the cell poles.

As will be discussed later, the “extrusion–

capture” model proposes a mechanism(s) that provides the force that “extrudes” the newly replicated origins from the replisome and “cap-tures” the origins at or near opposite cell poles.

As will also be discussed, for this to occur suc-cessfully, certain proteins must compact the chromosomes and guide the origin regions toward the cell poles. Eventually, the terminus is drawn into the replisome, where it becomes rep-licated; then the sister chromosomes separate, and segregation is completed as the duplicated termini remain near the cell center. As a result, newborn cells in slowly growing cultures of E.

coli, B. subtilis, and C. crescentus have a termi-nus near the new cell pole and an origin near the old cell pole (Fig. 3.17). Finally, the termi-nus in newborn cells is drawn toward the center of the cell by DNA replication in the centrally located replisome factory. There can be small differences among bacteria in the cellular loca-tion of chromosome regions. For example, in E. coli the origin regions localize at quarter-cell positions instead of at the cell poles, whereas in B. subtilis growing vegetatively or sporulating, they localize at the cell poles.

For a description of how the replisome as well as specifi c regions of the chromosome such as origins, termini, and regions between them, can be microscopically visualized, see note 35, as well as the references cited earlier concerning chromosome segregation in B. subtilis, E. coli, and C. crescentus. For more information about the location of the origins and termini, specifi -cally for fast-growing cells that initiate DNA

replication in the previous cell cycle, the student is referred to ref. 36.

There are important differences between the positioning of the replisome in E. coli and B. subtilis on the one hand, and C. crescentus on the other hand. These differences, which are related to differences in the cell cycle, are described later. (See Sections 3.1.7 and 3.1.8.) What moves the sister chromosomes apart, and what directs their movement?

Clearly, prokaryotic cells do not have a spindle apparatus. What then is responsible for the movement of sister chromosomes to opposite poles of the cell prior to cell division? Recently, there has been a great deal of research and much speculation. Several proteins have been implicated. Some of the proteins are thought to comprise the “motor” that moves the chro-mosomes, some of the proteins may attach the oriC regions to the opposite cell pole regions, and some of the proteins may compact the chromosomes and “guide” the chromosomes toward the opposite poles. Which proteins are these? We present an overview of current ideas, followed by a more detailed examination of the proteins thought to be involved in chromosome partitioning.

1. Sister chromosomes might be pushed out by the replisome to opposite halves of the cell

Because of the absence of a spindle apparatus similar to that found in eukaryotes, the mecha-nism for the partitioning of sister chromosomes toward opposite poles of the cell in prokaryotes is not well understood. Various models have been proposed. One model postulates the repli-some as a DNA-synthesizing “factory” that has motor proteins generating force that pushes the newly replicated sister chromosomes apart toward opposite cell poles. According to this model, referred to as the “extrusion–capture”

model (Fig. 3.17), in E. coli and B. subtilis the replisome is anchored to the membrane at the cell center; that unreplicated DNA enters the replisome and threads through it, whereupon the newly replicated sister chromosomes exit the replisome and partition toward opposite poles of the cell. In the slightly different situation of C. crescentus, (discussed later), the replisome is initially at the stalked pole of the cell, where

mechanism has been proposed whereby energy released by the combined action of the DNA polymerase and the DNA helicase, both of which can be considered to be force-generating motors, “pulls” the unreplicated DNA into the stationary replisome and “pushes” the replicated sister chromosomes, origin regions fi rst, out of the replisome into opposite halves

mechanism has been proposed whereby energy released by the combined action of the DNA polymerase and the DNA helicase, both of which can be considered to be force-generating motors, “pulls” the unreplicated DNA into the stationary replisome and “pushes” the replicated sister chromosomes, origin regions fi rst, out of the replisome into opposite halves