The bacterial genome is constantly subject to DNA damage, threatening its integrity and impacting cell survival. To counter this, bacteria have developed an array of sophisticated DNA repair mechanisms. Despite numerous genomic and molecular studies over the past five decades, one particular form of damage remains poorly understood – the formation and repair of single-stranded DNA gaps. We are aware of recombination-dependent and independent mechanisms of gap repair, however many of the players in these processes remain to be identified. In recent years it has been revealed DNA polymerase IV (Pol IV) among its other functions, participates in the recombination-dependent repair of DNA double-strand breaks. There are some obvious parallels in the recombination mechanisms of gap repair and break repair, raising the possibility that Pol IV also operates in gap repair. We are combining molecular and genetic approaches with advanced techniques for single-molecule live-cell imaging to investigating the potential role of Pol IV in gap repair.
We have carried out single-molecule live-cell imaging on E. coli cells that express a fluorescent protein fusion of Pol IV (DinB-YPet). We compared rates of focus formation, which is indicative of Pol IV working on DNA, in E. coli cells treated with azidothymidine (induces both gaps and breaks) and ciprofloxacin (induces breaks). Far more Pol IV foci are formed in cells treated with the gap-inducing compound azidothymidine than with ciprofloxacin, which does not induce gaps. Survival and sensitivity assays after treatment with azidothymidine and ciprofloxacin showed the deletion of Pol IV (and another polymerase, Pol V) drastically reduced the cells resistance to azidothymidine. These preliminary results suggest that Pol IV may be indeed involved in gap repair. To pinpoint the mechanism of repair, we are now investigating the genetic requirements underlying the formation of Pol IV foci in azidothymidine-treated cells.