Chapter 36 : Initiation and Termination of Chromosome Replication

Gene replication order map of B. subtilis as the basis for the circular genetic linkage map. (A) Chromosome depicted as a linear structure with replication starting at position 0 (origin) and proceeding sequentially through the markers (genes) indicated towards 1.0 (terminus) ( 94 ). (B) The same genes, with their order of replication (genes 1 through 10) on the Harford-Dedonder circular linkage map ( 54 ).

Autoradiographic visualization of the replicated portion of a reinitiated chromosome and suggestion of bidirectional replication to explain the result obtained. (A) The autoradiograph shows two smaller (reinitiated) loops contained within a larger loop. (B) Scheme for the formation of the structure seen by autoradiography from a circular chromosome containing a single oriC site (filled circle). Arrows indicate directions of movement of replication forks. The broken line represents the parental strand, and the solid line represents the newly synthesized one (taken from reference 120 ).

Conservation and variations of genes and their organization in the replication origin regions of abacterial chromosomes. Locations of ORFs (deduced from nucleotide sequences) are shown for three species. ORFs conserved between two or more species are indicated by various shadings. Open boxes are unique for one species. Sizes of ORFs are roughly to scale. Dotted lines in ORF boxes show the regions not yet sequenced. ORFs whose functions have not been identified by mutations are named either with ORF followed by the number of amino acids or with the molecular weight of the gene product. DnaA box regions are shown on a magnified scale with filled arrows for consensus boxes and open arrows for boxes different from the consensus by one base. The E. coli oriC region contains a region (dotted) of approximately 45 kb. Directions of transcription are indicated by solid arrows. Exceptions from the general rule are shown by dotted arrows.

Evolutionary relationships of replication origins. Hypothetical evolutionary relationships between three bacterial origins are shown schematically. The main filled arrow (dnaA), shaded arrows (other conserved genes flanking dnaA), and open arrow (gidA) show the organization and orientations of genes in the replication origin region. The numbers and orientations of DnaA boxes (small filled arrows) are arbitrary, showing that there are multiple repeats of DnaA boxes. The E. coli DnaA box region linked to dnaA is exceptional because there is only one DnaA box. Reprinted from Yoshikawa and Ogasawara ( 137 ) with permission.

Correlation between the cell's capacity for initiation and the amount of DnaA protein analyzed in a dnaA mutant cell. Cells of a dnaA1mutant of B. subtilis were grown in brain heart infusion medium for about six generations at 30°C, and then the culture was shifted to 49°C. At various times during incubation at 49°C, aliquots were shifted to 30°C in the presence of chloramphenicol (CP). At times indicated at both 49 and 30°C, purA/metB ratios of the chromosomal DNAs were determined. In parallel, the relative amounts of DnaA protein per unit volume of the culture incubated for indicated times at 49°C were determined by Western blotting (immunoblotting) using an anti-DnaA antibody. (A) Normalized purA/metB ratios are plotted against the time of incubation at 49°C (?) and at 30°C following incubation at 49°C for 15 min (?), 30 min (?), and 45 min (x). Bent arrows indicate times of shift-down to 30°C. (B) Data in panel A were replotted together with data for DnaA content of the cell. The purA/metB ratios at 150 min after temperature shift-down to 30°C are plotted against the incubation period at 49°C before the shift-down (?). The value for 60 min was taken from the data in a separate experiment. Datum points for DnaA content are averages of two experiments (?). Reprinted from Moriya et al. ( 81 ) with permission.

Functional elements in DnaA box regions of the B. subtilis chromosome. Structural elements that show various functions in initiation of chromosomal replication and its regulation are shown schematically. DnaA box regions are to scale, while the dnaA gene is reduced. Within the DnaA box regions, DnaA boxes are shown as filled vertical strips for consensus boxes and shaded vertical strips for boxes different from the consensus by one base. AT-rich 16-mers are indicated by triangles, and an AT-rich stretch is indicated by a shaded square. Promoters and directions of transcription are indicated by bent arrows. Regions responsible for inc activity and required for ars activity are indicated.

Model for control of the initiation cycle and for expression and function of DnaA protein. (A) DnaA box regions release DnaA protein temporarily after they are replicated. (B) dnaA gene expression is derepressed and actively transcribed to produce DnaA protein (?). (C) DnaA protein preferentially binds to the incB region of the DnaA box region to repress dnaA gene expression. (D) DnaA box regions form the initiation complex (initiosome); DnaB protein and binding to the cell membrane may be involved in this stage. Preformed DnaA protein now binds to all inc regions within at least two DnaA box regions to form the active oriC conformation. (E) Initiation and consequent replication of oriC take place. The relative sizes of the inc regions and the dnaA gene are as shown in Fig. 3 and 6 .

Sequence features of the terC region of the B. subtilis chromosome. (A) The region located at 174° on the physical map ( 51 ) and slightly offset in relation to the origin (oriC) comprises the gene for rtp and the upstream IRR, which is made up of IRI plus IRII. The clockwise-fork arrest site is shown as terC (see also Fig. 9 ). (B) Arrangement of sequences (ORFs) over a more extended chromosomal segment (-3 kb) spanning the terC region (IRR + rtp) ( 3 ). Strain W23 contains an additional ORF (ORF405) that strain 168 does not contain, which is inserted just left of the IRR. The gltC and gltA genes lie ∼1.5 kb to the right of the chromosomal segment sequenced, and the citM gene lies ∼90 kb to the left of it.

Putative RTP recognition sequences within the functional IRI of the B. subtilis chromosome. The two 8-bp segments (A and B, shaded) proposed as recognition sequences have been identified from the relative positionings of DNase I-protected regions and comparison of sequences within a total of 8 IRs. The heavy arrow defines IRI as originally described ( 22 ). The −10 region of the rtp promoter is located just to the left of A. The region of arrest of the leading strand of the clockwise replication fork is based on the work of Jannière and coworkers ( 20 ).

Model for clockwise-fork arrest and fork fusion within the terC region (IRR + rtp) of the B. subtilis chromosome. See text for explanation of model. ?, ?, binding sites for RTP; □, RTP; P, promoter.