To facilitate the measure, we inactivated abruptly a large quantity of RF in a population of synchronized cells. We chose to inactivate RF with Novobiocin, a drug that inhibits type II topoisomerases and mainly Gyrase, after establishing that priA2 cells were hypersensitive to Novobiocin. Gyrase eliminates the positive supercoils that accumulate in front of the RF and introduces negative supercoils ensuring the progression of the polymerase. The accumulation of positive supercoils in front of the RF, when Gyrase is inhibited, halts the progression of the polymerase and eventually inactivates RF. We measured by flow cytometry the accumulation of inactivated RF in different genetic backgrounds and found that dnaC2 priA2 cells accumulated times more arrested RF than isogenic priA cells at non-permissive temperature. This work led us to the identification of a new class of arrested RF – representing 60% of them, whose reactivation depends on PriA but apparently not on DnaC activity. Implications in terms of DnaC2 activity at nonpermissive temperature and in terms of the frequency of RF inactivation during normal growth are discussed. To shed light on this matter, we developed a method allowing us to measure the fraction of cells with active RF within a population. Synchronized cells that initiated and completed a round of replication accumulate within the peak at 2 genomes per cell, while those in which replication did not Norethindrone initiate accumulate within the peak at one genome per cell. Cells were arrested before completion of replication and not reactivated accumulate in the valley between 1 and 2 genomes per cell. The drift of the peak on DNA histograms of cells harvested before, the temperature downshift is almost negligible. Therefore, cells in which both RF were arrested within 10 minutes after the initiation of replication and not reactivated, accumulate with those that did not initiate replication. Hence, and in order to assess the ability of dnaC2 cells to reactivate arrested RFa large proportion of RF was transiently inactivated soon after initiation of replication in a synchronized population of cells. Then, the cells were brought back to growth conditions permissive with respect to replication. Under such experimental conditions, cells in which replication did not initiate and those in which both RF were arrested – during the inactivation procedure – and not reactivated, accumulate within the peak at one genome d., We turned to the analysis of cytograms, in which the DNA content is plotted over the FSC, which gives a rough estimate of cell mass,to delineate more precisely the fraction of cells with one genome. Given this starting situation, the proportion of cells undergoing replication under a given condition can be extracted from the fraction of cells with one genome at t0 and t40. The rationale behind this experiment was based on the requirement of the helicase activity specified by PriA and the following recruitment of the primosomal proteins for the reactivation of arrested RF. Another pathway �C driven by PriC and independent of PriA �C was deduced from the synthetic lethality associated with a double mutant priA priC. Yet, the absence of phenotype attributable to a priC single mutant led to the assumption that the PriA-driven mechanism was the major RF reactivation pathway. Is DnaC systematically required to reactivate arrested RF? If DnaB were still present on arrested RF, for example, its reactivation should not require DnaC. Such a Amantadine hydrochloride situation should not require PriA either, because the very function of PriA is to assist the loading of the replicative helicase onto DNA. Thus, this hypothesis may be excluded. We may instead consider that DnaC2 is active for reloading DnaB at arrested RF and at nonpermissive temperature.