## 3 Discussion  
In response to a replication stress, a stalled fork can be remodeled, but the function(s) that process(es) a stalled fork and the molecular basis of its regulation are poorly characterized in bacteria other than those of the \(\gamma\)-Proteobacteria class. Genetic analysis showed that when the single genome of an inert mature _B. subtilis_ spore is exposed to ionizing radiation and then the spores are revived under unperturbed conditions, RecA, RecG, RuvAB, RecU, and DisA are required for survival, but neither RecQ-like (RecS and RecQ) remodelers nor the end-resection functions (AddAB and RecJ) are involved in spore revival [12; 13]. Cytological studies have shown that DisA scans the chromosome and pauses at RecA bound to lesion-containing gaps at stalled replication forks rather than at DSBs during sporulation [17; 18]. Dynamic DisA also pauses in unperturbed exponentially growing \(\Delta\)_recU_ cells but not in the exponentially growing \(wt\) cells [23]. In the presence of MMS- or the UV mimetic 4-nitroquinoline 1-oxide (4 NQO)-induced lesions, RecA filamented on the ssDNA gaps triggers the DNA damage response and stops DisA scanning, perhaps loading it at stalled or reversed forks [18]. DisA bound to branched intermediates reduces c-di-AMP synthesis to levels comparable to that in the absence of DisA, in otherwise \(wt\) cells [21]. Low c-di-AMP levels indirectly inhibit cell proliferation (see the introduction). Taking these data into account, we hypothesized that DisA could be providing, together with fork remodelers, a mechanism to cope with replicative stress and could protect stalled forks. Previously, it has been shown that during replication stress, DisA limits the activities of RecA and RecG [18; 27]. Here, we show that DisA interacts with RuvB and inhibits the DNA-dependent ATPase activity of the RuvAB complex (Figures 2A,B and 4). RuvAB fails to reverse a stalled fork at protein concentrations that efficiently regress a reversed fork, and DisA cannot stimulate the conversion of a stalled into a reversed fork (Figure 3). It is likely that during a replication stress, RecG (or RecA) converts a stalled fork into a reversed fork [27]. The reversed fork may be further processed by the RuvAB-RecU resolvasome to yield a one-ended DSB and a nicked duplex, as reported in _E. coli_[10; 28; 29]. However, if this occurs, these physiological reversed forks intermediates would become a pathological structure, as during the early stages of spore revival (see the introduction). We show here that DisA limits the processing of a reversed fork by the RuvAB-RecU resolvasome (Figures 3 and 5), and accordingly, it partially reduces chromosomal breakage of unperturbed growing cells (Figures 2F and 7). From the results presented here and in previous reports, we propose that, in response to a 4 NQO- or MMS-induced insult, the repli  
Figure 7: Proposed DisA mode of action in the presence of RecU and RuvAB. An unrepaired DNA lesion on the leading-strand template (green square) causes blockage of replication fork movement. Fork reversal by the RecA or RecG remodeler may form an HJ DNA structure and this process is inhibited by DisA. In the upper panel, DisA suppresses fork reversal and HJ resolution by the RuvAB-RecU resolvasome (**a,b**), avoiding the formation of a one-ended DSB. In the lower panel, DNA synthesis extends the regressed fork to convert it into an HJ-like structure, and fork restoration is catalyzed either by RuvAB or RecG (**c,d**), followed by damage removal by specific repair mechanisms. This process is also downregulated by DisA. DisA bound to HJ DNA decreases c-di-AMP synthesis, indirectly increasing (p)ppGpp synthesis and inhibiting cell proliferation. strand degradation and genome instability. In other words, DisA bound to a reversed fork protects the extruded nascent strands from an unscheduled cleavage by the RuvAB-RecU resolvasome until the lesion is circumvented (Figures 2, 4 and 7a,b).