# DisA Limits RecG Activities at Stalled or Reversed Replication Forks
## 1 Introduction
subtilis_ origin). The diadenylate cyclase (DAC) DisA converts a pair of ATP molecules into cyclic 3\({}^{\prime}\), 5\({}^{\prime}\)-diadenosine monophosphate (c-di-AMP) [21], an essential second messenger that regulates a wide variety of physiological functions, [22], including the monitorization of replication perturbations. Single-cell analyses revealed that DisA, acting as a DNA damage checkpoint, shows a dynamic movement along the chromosome during sporulation, but it pauses upon DNA damage until the offending lesion is removed [23]. DisA pauses at a cognate site formed at or downstream RecA engagement to DNA lesions that compromise replisome progress [24]. Paused DisA drops the synthesis of c-di-AMP to levels comparable to that in the absence of DisA in response to replication perturbations, as those induced by methyl methane sulfonate (MMS) [25; 26]. Indeed, DisA binding to DNA substrates that mimic branched intermediates suppresses c-di-AMP synthesis, but not when bound to double-stranded (ds) DNA [21; 27], placing the primary function of DisA at a stalled or reversed fork to circumvent or bypass offending lesions. In this context, it has also been observed that: (i) in unperturbed exponentially growing cells, DisA shows a dynamic movement in wt cells, but it becomes static in the absence of the RecG branch migration translocae (where branched recombination intermediates accumulate) [27]; and (ii) the _disA_ and _recG_ genes show an epistatic interaction upon DNA damage [27]. The RecG translocae is ubiquitous among Bacteria, land plants, green algae and moss (targeted to mitochondria and chloroplast) [28; 29]. RecG homologues have not been detected in archaea and other eukaryotes. Here, the mammalian SMARCAL1 translocase may be its functional ortholog [30]. RecG is a multi-functional protein and there is conflicting evidence on its mode of action [29]. In vitro, _E. coli_ RecG (RecG\({}_{Eco}\)) promotes branch migration of a stalled replication fork to form a HJ-like structure, and it can also branch migrate the resulting HJ back to a fork structure (fork restoration), as the SMARCAL1 enzyme does [30; 31; 32; 33; 34]. In the presence of a reconstituted replication system, it is observed that the reversed forks formed by RecG\({}_{Eco}\), rather than by RuvAB\({}_{Eco}\), are the substrate for RuvC\({}_{Eco}\) (counterpart of _B. subtilis_ RecU) cleavage [35]. Other studies proposed a division of labor with RecG\({}_{Eco}\) acting at DSBs, and the RuvAB\({}_{Eco}\) translocase acting at stalled forks reviewed in [7]. The role of RecG in other bacteria remains poorly understood because not all the activities associated to RecG\({}_{Eco}\) are present in other distant RecG proteins [29]. For instance, in vivo, inactivation of _recG_ shows disparate phenotypes in the different bacteria tested: (i) a _Helicobacter pylori_\(\Delta\)_ruvAB_\(\Delta\)_recG_ mutant strain shows no significant difference with the wt control in response to DNA damage [36]; (ii) an _E. coli_\(\Delta\)_ruvAB_\(\Delta\)_recG_ mutant strain has a severe DNA repair defect [37]; and (iii) a _\(\Delta\)_recG_ mutation is synthetically lethal in the \(\Delta\)_ruvAB_ context in _B. subtilis_ and _Neisseria gonorrhoeae_[38; 39]. Finally, there are biochemical activities associated with RecG\({}_{Eco}\) that are not observed in _B. subtilis_: (i) RecG\({}_{Eco}\) binds and unwinds a variety of branched DNA substrates, including HJs, D-loops and R-loops, but unwinding of R-loops was not seen with _B. subtilis_ RecG [40]; and (ii) RadD\({}_{Eco}\) bound to abandoned fork structures modulates RecG\({}_{Eco}\) activities [41], but a RadD-like enzyme is absent in _B. subtilis_ cells. These observations suggest that both RecG enzymes may work at forked DNA structures, but the information obtained with RecG\({}_{Eco}\) may not be easily extrapolated to other bacteria, highlighting the vital importance of revision in science.