The crystal structure of the Escherichia coli replication-terminator protein (Tus) bound to terminus-site (Ter) DNA has been determined at 2.7 A resolution. The Tus protein folds into a previously undescribed architecture divided into two domains by a central basic cleft. This cleft accommodates locally deformed B-form Ter DNA and makes extensive contacts with the major groove, mainly through two interdomain beta-strands. The unusual structural features of this complex may explain how the replication fork is halted in only one direction.

MukF, MukE and MukB proteins form a complex that may participate in the organization of folded sister chromosomes in Escherichia coli. We have found that a MukB-GFPuv4 fusion protein is observed as discrete fluorescent foci, which are localized within cellular spaces occupied by nucleoids, but not at the constriction site of cell division in living cells. In contrast, MukB-GFPuv4 is distributed throughout the whole cell when either MukF or MukE is absent. Statistical analysis revealed that most newborn cells have two foci of mukB-gfpUV4 at one-quarter and three-quarter positions in the cell length and one focus of SeqA-bound nascent DNA at or near the middle of the cell. Subsequently, the single SeqA focus divides into two foci, and then these migrate to the one-quarter and three-quarter positions. Before cell division, most long cells have two SeqA foci and four MukB-GFPuv4 foci. In early stationary phase, SeqA foci disappear, but one or two foci of MukB-GFPuv4 remain. We discuss the reorganization and proper arrangement of folded sister chromosome in the cell quarter positions, which are performed after release from the long-time cohesion of sister chromosomes.

Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic k-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase-and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos-the cis-element required for linearization-was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.

SummaryTo demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated. After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome. In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay. These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated. Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus. This supports the translocating replication apparatuses model.

The ␤-subunit of DNA polymerase III is located as one or two condensed clusters within the nucleoidoccupied space in exponentially growing cells of Escherichia coli. When chromosome replication is terminated after incubation at nonpermissive temperature in a temperature-sensitive dnaC mutant, the ␤-subunit is located in the cytosolic spaces of the cell poles.The dnaN gene in Escherichia coli encodes the ␤-subunit of DNA polymerase III. A dimer of the ␤-subunit forms the DNA sliding clamp. The dimer is loaded to DNA in an ATP-dependent manner catalyzed by the ␥ complex (a subassembly of DNA polymerase III) to form the preinitiation complex. In the next step, the DNA polymerase III core that catalyzes DNA synthesis associates with the preinitiation complex to form the initiation complex. The ␤-subunit exists as 300 to 5,000 dimers per cell (for reviews, see references 1, 6, 7, and 9). In this work, we have analyzed the subcellular localization of the ␤-subunit in exponentially growing wild-type cells and temperature-sensitive dnaC mutant cells synchronized for initiation of chromosome replication.Subcellular localization of the ␤-subunit of DNA polymerase III and SeqA in exponentially growing cells. We analyzed the subcellular localization of the ␤-subunit of DNA polymerase III by indirect immunofluorescence microscopy (4) using rabbit anti-␤-subunit polyclonal antibody. In exponentially growing cells of strain YK1100 (a tryptophan-deficient mutant derived from W3110 [11]) at 37°C in M9 glucose medium supplemented with L-tryptophan (50 g/ml), the ␤-subunit formed one or two distinct condensed clusters within the nucleoid. Besides clear clusters, a portion of ␤-subunit molecules was distributed in polar cytosolic spaces in some cells (Fig. 1A and Table 1). The average length of cells with two ␤-subunit clusters was longer than that of cells with one ␤-subunit cluster (Table 1 and (Table 1 and Fig. 2A). This result eliminates the possibility that this type of cells without clusters was in a specific stage of the cell cycle, for example, the D period (the nonreplication period under the slow-growth conditions at a 55-to 60-min doubling time). Presumably, it is difficult to detect faint clusters, because of a high background of ␤-subunit dispersed throughout the whole cell.In a rich medium (L medium), cells had one, two, three, and four clusters of the ␤-subunit (Table 1) (Table 1). The average length (3.06 Ϯ 0.86 m) of cells without clear clusters was similar to that (3.21 Ϯ 0.85 m) of total cells with one, two, three, and four clusters (Table 1 and Fig. 2B). It is unlikely that this type of cells without clusters was in a specific stage of the cell cycle.We previously reported that SeqA is localized as discrete foci in growing YK1100 cells. SeqA forms clusters with hemimethylated nascent DNA segments behind replication forks (4, 10; for a review, see reference 3). We therefore analyzed the subcellular localization of the SeqA protein as a landmark of hemimethylated nascent DNA segments in the cell cycle. As shown in...