Replication of bacterial chromosomes is not a continuous process, but is occasionally impaired by the encounter with obstacles such as DNA lesions, secondary structures or DNA bound proteins. When replication arrest causes the disassembly of the replication machinery, replication needs to be re-initiated from a non-origin sequence. Replication restart, in such cases, requires the loading of the DnaB helicase on the inactivated replication fork, which, in turn, triggers the binding of the DnaG primase and of the holoenzyme polymerase III (Pol III HE). DnaB is loaded by a specific protein called DnaC, which is targeted to the inactivated replication fork by the preprimosome proteins PriA, PriB, PriC and DnaT (reviewed in; Marians, 2000; Sandler and Marians, 2000). The assembly of the primosome involves (i) recognition of a forked structure or of a D-loop by the PriA protein; (ii) assembly of the pre-primosome proteins; and (iii) loading of DnaB by the DnaC–DnaB complex on the single-strand region of the lagging strand template, coated by single-strand DNA binding protein (SSB). PriA is the key protein for replication restart, presumably because of its targeting function. The absence of PriA leads to poor growth, SOS induction, cell filamentation and recombination deficiency (Kogoma et al., 1996; Sandler et al., 1996). In contrast, inactivation of either priB or priC genes causes no apparent deleterious phenotype. PriB and PriC proteins were proposed to fulfill redundant functions for PriA-dependent primosome assembly, based on the observation that although inactivation of either of them has no deleterious effect, inactivation of both is lethal (Sandler et al., 1999). However, purified PriB and PriC proteins were not interchangeable in vitro, as replication initiation from a recombination intermediate was strictly dependent on PriB (Liu and Marians, 1999; Liu et al., 1999). The reasons for the difference between in vivo and in vitro observations remained to be understood.
The residual viability of the priA null mutant relies on the presence of the PriC protein, suggesting that in the absence of PriA, DnaC can be targeted to arrested forks by a weak PriC-dependent pathway (Sandler, 2000). The efficiency of the PriC pathway was greatly enhanced by gain-of-function mutations in dnaC such as the dnaC809 mutation, which restores a wild-type phenotype to priA null mutants (Sandler et al., 1996). Finally, all preprimosome proteins were dispensable in strains carrying a further improved dnaC allele with two mutations, called dnaC809 820 (Sandler et al., 1999). In vitro studies confirmed that dnaC809 mutation conferred a gain of function. Indeed, the DnaC809 protein could promote in vitro DnaB loading and replication initiation from a recombination intermediate in the absence of all PriA, PriB and PriC proteins (Liu et al., 1999; Xu and Marians, 2000). In vivo, dnaC809 mutation only partially suppresses the growth defect of a triple priA priB priC mutant whereas dnaC809 820 mutation is able to suppress almost completely the need for PriA, PriB and PriC proteins (Sandler et al., 1999). The lethality of the priA priC double mutant suggested that replication restart is a process essential for viability (reviewed in Sandler and Marians, 2000). However, flow cytometry analysis in a dnaCts mutant suggested that replication arrest might be less common than previously thought, as 80% of the chromosomes can replicate to completion in cells deficient for replication restart (Maisnier-Patin et al., 2001). Consequently, in wild-type cells the importance of certain pre-primosome proteins may be underestimated because of a limited need for replication restart in the absence of a specific replication defect.
Here, we used the holDG10 mutation to analyse the requirement for pre-primosome proteins in a mutant cell in which the frequency of replication arrest is increased. holD encodes the Psi subunit of Pol III, a subunit of the Gamma complex involved in the loading of the processivity clamp at the onset of Okazaki fragment initiation (Naktinis et al., 1996; Yuzhakov et al., 1999). In the holDG10 mutant, replication is frequently arrested, leading to a specific reaction called replication fork reversal (Flores et al., 2001). This reaction involves the annealing of leading and lagging strand upon replication arrest, forming a double-strand end and a Holliday junction (Fig. 1, first step). The Holliday junction is recognized by RuvAB. The double-strand end is acted upon by RecBCD and is either re-incorporated into the chromosome by RecA- RecBCD-dependent homologous recombination (Fig. 1A), or degraded by the exonuclease V action of the enzyme (Fig. 1B). In the absence of RecBCD, resolution of the Holliday junction formed by replication fork reversal leads to chromosome breakage (Fig. 1C; Seigneur et al., 1998; reviewed in Michel, 2000; Michel et al., 2001). The replication fork reversal model is supported in the holDG10 mutant by two sets of data: (i) direct analysis of chromosomes showed RuvABC-dependent linearization in the absence of RecBCD (Fig. 1C) and (ii) genetic analysis showed that the holDG10 mutant requires RecBCD for viability, whereas it only requires RecA in the absence of exonuclease V and vice versa (Fig. 1A and B). In the present work, we tested the importance of PriA, PriB and PriC proteins for the viability of the holDG10 mutant. We show that PriA is essential, PriB important and PriC dispensable for replication restart in the holD strain. Therefore, in a strain with increased frequency of replication arrest, PriB is more important for replication restart than PriC, as in the in vitro reconstituted reaction. We also tested the effects of the dnaC809 and dnaC809 820 allele on cell viability. The dnaC809 allele only partly restored the viability of holD priA or holD priB mutant, whereas dnaC809 820 allele restored full viability. Finally, we show that the priA300 allele, which inactivates only the helicase function of PriA in vitro, affects the viability of the holDG10 mutant.