This chapter discusses the components of the terminus-region replication fork-arrest system, their organization and structural features, and their functioning during termination of a round of vegetative replication in Bacillus subtilis. Progress in characterizing and understanding the components and features associated with termination of replication in B. subtilis and Escherichia coli proceeded almost in parallel. The current status of the dif system in B. subtilis is discussed in this chapter. There is emerging evidence to suggest that the replication fork trap might enable more efficient operation of this system. The basis for this will become apparent in the discussion of the various components and features associated with their operation. The functional fork-arrest complex contains two dimers of replication terminator protein (RTP) bound to overlapping A and B sites within the 30-bp terminator. Successful partitioning of replicated daughter chromosomes, generated upon completion of the termination phase of replication, requires the formation of completely separable chromosomes. The existence of multiple sequences of ~8 nt in length with a skewed distribution between the two strands of each oriC-terminus arm (replichore) of the chromosome has more recently been uncovered, and it is now clear that the polarity switch point for these sequences within the terminus region of E. coli coincides with dif. Finally, the chapter focuses on replication fork arrest near oriC under stringent conditions.

Features of the terminus region of the B. subtilis 168 chromosome. The position and orientation of each of the nine replication terminators are shown diagrammatically in the upper section, which covers the ∼150 to 190° segment of the chromosome. TerI, TerIII, TerV, and TerIX are oriented to block the clockwise replication fork; TerII, TerVIII, TerIV, TerVII, and TerVI are oriented to block the counterclockwise fork. In the majority of replicating chromosomes, the two forks meet in the close vicinity of TerI (see text). The shaded region corresponds to the segment occupied by the SPβ prophage, with TerIX lying within the prophage. The positions of attR (for SPβ) and a number of genes and features associated with replication fork arrest and daughter chromosome resolution are also shown. The dif site is at ∼166° (48a). The lower sections provide more detailed information on the location of the terminators. The nucleotide positions are for nt 1 of the terminator (see Fig. 2) and have been rounded to the nearest 100 nt. The lower right section shows the position and orientation of each terminator (AB) in relation to flanking genes or unidentified reading frames. A and B correspond to the A site and B site for RTP binding in each case (see Fig. 2), with the shaded regions corresponding to the SPβ prophage. The positions of rho-independent transcription terminators are indicated by the conventional stemmed circles.

10.1128/9781555817992/fig8-1_thmb.gif

10.1128/9781555817992/fig8-1.gif

FIGURE 1

Features of the terminus region of the B. subtilis 168 chromosome. The position and orientation of each of the nine replication terminators are shown diagrammatically in the upper section, which covers the ∼150 to 190° segment of the chromosome. TerI, TerIII, TerV, and TerIX are oriented to block the clockwise replication fork; TerII, TerVIII, TerIV, TerVII, and TerVI are oriented to block the counterclockwise fork. In the majority of replicating chromosomes, the two forks meet in the close vicinity of TerI (see text). The shaded region corresponds to the segment occupied by the SPβ prophage, with TerIX lying within the prophage. The positions of attR (for SPβ) and a number of genes and features associated with replication fork arrest and daughter chromosome resolution are also shown. The dif site is at ∼166° (48a). The lower sections provide more detailed information on the location of the terminators. The nucleotide positions are for nt 1 of the terminator (see Fig. 2) and have been rounded to the nearest 100 nt. The lower right section shows the position and orientation of each terminator (AB) in relation to flanking genes or unidentified reading frames. A and B correspond to the A site and B site for RTP binding in each case (see Fig. 2), with the shaded regions corresponding to the SPβ prophage. The positions of rho-independent transcription terminators are indicated by the conventional stemmed circles.

(A) A ribbon diagram of a dimer of RTP with the two α3 helices and the two β2 strands (“wing” regions) as well as the B terminus identified (modified from reference 7). (В) A model showing the positioning of two dimers of RTP on the DNA when bound to adjacent binding sites as present in a complete terminator (based on the positioning of RTP bound to a single site [60a]). Amino acid residues for both dimers (shaded light and dark) are shown as space-filling models. Interaction between the two dimers positioned on approximately opposite sides of the DNA helix is possible because part of the “winged-helix” domain protrudes from the DNA on the same side for both dimers. The images were generated using Molscript (26).

10.1128/9781555817992/fig8-3_thmb.gif

10.1128/9781555817992/fig8-3.gif

FIGURE 3

(A) A ribbon diagram of a dimer of RTP with the two α3 helices and the two β2 strands (“wing” regions) as well as the B terminus identified (modified from reference 7). (В) A model showing the positioning of two dimers of RTP on the DNA when bound to adjacent binding sites as present in a complete terminator (based on the positioning of RTP bound to a single site [60a]). Amino acid residues for both dimers (shaded light and dark) are shown as space-filling models. Interaction between the two dimers positioned on approximately opposite sides of the DNA helix is possible because part of the “winged-helix” domain protrudes from the DNA on the same side for both dimers. The images were generated using Molscript (26).

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26.Kraulis, P. J.1991. Molscript: a program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst.24:946–950.

27.Kuempel, P.,, A.Hogaard,, M.Neilsen,, O.Nagappan,, and M.Tecklenburg. 1996. Use of a transposon (Tn dif) to obtain suppressing and nonsuppressing insertions of the dif resolvase site of Escherichia coli.Genes Dev.10:1162–1171.

44.Pérals, K.,, F.Cornet,, Y.Merlet,, I.Delon,, and J.-M.Louarn. 2000. Functional polarization of the Escherichia coli chromosome terminus: the dif site acts in chromosome dimer resolution only when located between long stretches of opposite polarity. Mol. Microbiol.36:33–43.