A Defined Terminal Region of the E. coli Chromosome Shows Late Segregation and High FtsK Activity

Background: The FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV. Methodology: We have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a,400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPSrecognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions. Significance: Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last step

Xer Recombinase and Genome Integrity in Helicobacter pylori, a Pathogen without Topoisomerase IV

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis

DNA Replication Fork Arrest by the Bacillus subtilis RTP-DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP-DNA Binding

In order to elucidate the mechanism of DNA replication fork arrest by the replication terminator protein (RTP)-DNA complex, a set of RTP fusion proteins were constructed in which peptides of various sizes were fused to the C terminus; this placed the peptides at a surface location that was predicted to come into contact with the DNA replication machinery during fork arrest. The fusion proteins were capable of replication fork arrest in vivo, but they had a significantly reduced efficiency compared to wild-type RTP, which was not directly proportional to peptide size or sequence. Importantly, the fusion proteins retained completely normal RTP-DNA binding affinity. These findings rule out the molecular clamp model as the sole explanation for fork arrest by RTP, and suggest that RTP interacts with the replication machinery in a manner that directly contributes to the fork arrest mechanism. Crown Copyright © 2006

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Tev sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. Ln support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the Rm-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork. (C) 1999 Academic Press

Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius

The “baby machine” provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at ≈88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of the MCM helicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase