DNA damage contributes to genome instability by creating barriers that hinder the progression of the replication machinery (replisome) during DNA replication . Consequently, DNA replication forks that stall or collapse due to encounters of the replisome with DNA damage must be reactivated to allow complete replication of the genome and ensure survival of the cell. DNA replication restart pathways provide bacterial cells with a mechanism to reactivate replisomes that are disrupted in this manner . Catalyzed by primosome proteins such as PriA, PriB, PriC, DnaT, and DnaG, DNA replication restart pathways facilitate origin-independent reloading of the replicative helicase onto a repaired DNA replication fork in a process that involves coordinated protein and nucleic acid binding within a nucleoprotein complex called the DNA replication restart primosome .
DNA replication restart initiated by PriA helicase is a highly coordinated process and probably represents the major pathway of DNA replication restart in E. coli. PriA belongs to the DExH family of DNA helicases and is well-conserved among sequenced bacterial genomes . PriA is thought to recognize and bind to repaired DNA replication forks and D-loop recombination intermediates, facilitate assembly of the primosome complex by recruiting other primosome proteins, and catalyze duplex DNA unwinding using energy furnished by hydrolysis of ATP [4, 5]. Recruitment of PriB to a PriA:DNA complex stabilizes PriA on the DNA  and enhances its helicase activity through a mechanism that involves PriB's single-stranded DNA-binding activity . Formation of a PriA:PriB:DNA complex leads to recruitment of DnaT, perhaps through physical interactions with PriB . The function of DnaT is not well understood, but it has been proposed that DnaT binding leads to dissociation of single-stranded DNA (ssDNA) from PriB through a competition mechanism, possibly exposing the ssDNA on the lagging strand template for reloading the replicative helicase, which ultimately leads to fork reactivation .
While studies of DNA replication restart pathways have focused primarily on the well-studied E. coli model organism, DNA replication restart has been shown to be important in other bacteria as well, including the medically important bacterium, Neisseria gonorrhoeae. N. gonorrhoeae is a gram-negative bacterium and the causative agent of gonorrhea. Infections are associated with a host inflammatory response that is mounted against the pathogen involving phagocytic cells such as polymorphonuclear granulocytes . The ability of phagocytes to produce reactive oxygen species as an antimicrobial mechanism has been well-established, and commensal organisms such as lactobacillus species have been shown to produce and secrete H2O2, thus making it likely that N. gonorrhoeae faces considerable oxidative challenges in infected individuals [10, 11].
A variety of studies have examined the sensitivity of N. gonorrhoeae to oxidative stress. Among them, one has demonstrated that N. gonorrhoeae can utilize enzymatic mechanisms such as catalase, peroxidase, and glutathione to protect against reactive oxygen species , another has shown that manganese is important for chemically scavenging superoxide , and yet another has revealed a role for DNA recombination and repair enzymes such as RecA, RecBCD, and enzymes of the RecF-like pathway in resistance to oxidative stress . In addition, PriA has been shown to play a critical role in DNA repair and in resisting the toxic effects of oxidative damaging agents, suggesting that DNA replication restart pathways might play an important role in N. gonorrhoeae resistance to oxidative stress and overall pathogenicity . This notion is further supported by observations that priA from a related bacterium, Neisseria meningitidis, is an important virulence determinant and is important for resisting oxidative injury and promoting bacterial replication .
Studies of DNA replication restart pathways in diverse bacteria such as E. coli and N. gonorrhoeae have revealed species differences in the composition of the DNA replication restart primosome and in the functions of the individual primosome proteins. For example, N. gonorrhoeae lacks a recognizable homolog of dnaT in its genome, suggesting that the N. gonorrhoeae PriA-PriB pathway might be significantly different from the E. coli PriA-PriB-DnaT pathway. Furthermore, physical interactions between primosome components show variation in their individual binary affinities: the physical interaction between PriA and PriB is rather weak in E. coli, but relatively strong in N. gonorrhoeae, and the physical interaction between PriB and ssDNA is strong in E. coli, but relatively weak in N. gonorrhoeae [8, 17, 18]. Thus, the affinities of binary interactions between primosome components are reversed between the two species.
Since the ssDNA-binding activity of PriB is important for PriB-stimulation of PriA's helicase activity in E. coli , there might be significant functional consequences for the variation in affinities of physical interactions within the N. gonorrhoeae PriA-PriB primosome. In this study, we investigated the functional consequences of the affinity reversal phenomenon by examining the helicase activity of N. gonorrhoeae PriA, and we determined how PriA-catalyzed ATP hydrolysis and DNA unwinding are affected by N. gonorrhoeae PriB.