The replication checkpoint control in Bacillus subtilis: identification of a novel RTP‐binding sequence essential for the replication fork arrest after induction of the stringent response

Abstract

SummaryWe have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (¹128 kb from oriC ). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (BЈ-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This BЈ-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.

A Replication Terminus Located at or Near a Replication Checkpoint of Bacillus subtilis Functions Independently of Stringent Control

“…We have also examined the replication intermediates from the same plasmid containing the L1 core sequence by onedimensional gels and found no evidence of replication arrest, consistent with earlier published results (Ref. 8 and data not shown).…”

“…Perhaps the explanation for the difference is that only the 17-bp core sequence of L1 was used by them for RTP binding experiments and the binding behavior of the complete core and auxiliary sequence was not examined (8). It is, however, more difficult to explain as to why stringent response-dependent conditional arrest of replication was observed by the other group in a genomic DNA fragment that included L1 (8). We saw no evidence that replication arrest at L1 was modulated by stringent response.…”

“…The sequence bound to a single dimer of RTP and was reported to have arrested replication in a polar fashion under stringent but not under relaxed conditions (8). This result was surprising because two interacting dimers of RTP, bound to the core and auxiliary site are known to be essential for replication termination (9 -11) and in fact polarity is generated from symmetrical dimers of RTP (12) by differential interaction with the core and auxiliary sites of Ter (13).…”

Replication Fork Stalling at Natural Impediments

“…This phenomenon was called "replication checkpoint of B. subtilis," and its functioning was shown to depend on the presence of RTP (156); however, the mechanism of regulation of the replication arrest is not known. The RTP binding site was located around one of the two arrest sites (4). It was further shown that this site was bipartite, like Ter, and bound two RTP dimers (although much more weakly than Ter sites from the terminus).…”

The extrusion-capture model for chromosome partitioning in bacteria

“…The replication arrest experiments took advantage of elegant work characterizing the effects of amino acid starvation on DNA replication. On amino acid starvation (induction of the stringent response), a reversible DNA replication arrest occurs in B. subtilis in regions ∼100-130 kbp to the left (LSTer, left stringent terminus region) and ∼150-200 kbp to the right (RSTer) of the origin of replication (Levine et al 1991(Levine et al , 1995Autret et al 1999). Replication resumes in the STer regions when amino acids are added back to the cells.…”