# DisA Limits RecG Activities at Stalled or Reversed Replication Forks
## 4 Discussion  
Our work provides new insights into a comprehensive model of DisA and RecG functions contributing to genome integrity by stabilizing stalled or reversed replication forks. In _E. coli_, four proteins (RecA, RecG, RuvAB and RecQ) may process stalled or reversed forks [9; 10; 11; 59]. However, when the single genome of an inert mature haploid _B. subtilis_ spore is damaged, unperturbed spore revival requires RecA, RecG and RuvAB, but not a RecQ-like DNA helicase (RecS or RecQ) [17; 20]. Furthermore, although it is poorly known if these enzymes act in coordination with each other, the _disA_ gene is epistatic to the _recA_, _recG_ or _ruvAB_ gene in response to DNA damage [20]. The role of DisA in limiting RecA -mediated DNA strand exchange was previously reported [43]. Here, we report the interplay of DisA with RecG, and how RuvAB and DisA might contribute to replication fork remodeling and replication restart will be addressed elsewhere. We are aware that the phenotypes seen in cells lacking RecG are very complex [29; 60], and that DisA only contributes to the repair of perturbed replication forks [20; 27]. In fact, the inactivation of _B. subtilis recG_ renders cells extremely sensitive to DNA damaging agents that promote single-strand gaps and DSBs [60], while the absence of DisA renders cells only sensitive to single-strand gaps, but they remain recombination proficient and are apparently as capable of repairing DSBs as the wt control [26; 27], suggesting that DisA might only react to replication stress. However, it seems that although RecG may have other functions in which DisA is not involved, its roles upon replication stress are controlled by DisA. RecG\({}_{\text{Eo}}\) and RecG are evolutionarily separated by more than 1500 million years. _In vitro_, both enzymes, as well as mammalian SMARCAL1, unwind a stalled fork to form a HJ-like structure (fork reversal) and regress a HJ DNA leading to fork restoration (Figures 2 and 3) [10; 11; 30; 34; 56]. Thus, it is likely that RecG-mediated replication fork remodeling is one of its genuine in vivo activities, well conserved in evolution. In this work, we observed that DisA bound to the branched intermediate limits both RecG activities at stalled and reversed forks (Figure 3). The effect of DisA seems to be specific because DisA neither affects PriA-dependent replication initiation, nor DNA replication elongation [20]. To confirm this observation, we have shown here that DisA neither affects PrcA-mediated ATP hydrolysis nor unwinding of tailed HJ DNA substrates (Supplementary Figure S3), suggesting that DisA does not act as a protein roadblock that inhibits RecG activities. DisA monitors genomic stability by forming a rapid-moving focus on the nucleoid [23]. Upon perturbation of DNA replication, DisA pauses in a RecO- and RecA-dependent manner, perhaps at stalled replication forks [24]. This is consistent with the observation that, upon UV treatment, >80% of the RecA foci co-localized with the replisome [61] rather than with the lesion-containing gap left behind by the replisome. Here, RecA interacts with and loads DisA at branched DNA (stalled or reversed forks), as observed in vitro [24]. DisA bound to HJ DNA drops the synthesis of c-di-AMP and this inhibition is additive in the presence of RecG (Figure 1C), although these proteins do not form a stable complex (Supplementary Figure S2A,C). These results suggest that DisA transiently interacts with RecG at a HJ intermediate, with a subsequent blockage of DisA-mediated c-di-AMP synthesis to provide a fail-safe mechanism that avoids the uncoupling of the cell cycle. Low c-di-AMP levels indirectly increase the production of (p)ppGpp [52; 53], that in turn inhibits DNA primase [54], blocking cell proliferation (Figure 5). Our results suggest that DisA, which is absent in _E.