Role of the Rep Helicase Gene in Homologous Recombination in Neisseria gonorrhoeae

Abstract

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.Neisseria gonorrhoeae (gonococcus [Gc]) is an obligate human pathogen that possesses a number of mechanisms that have enabled it to persist within human populations, including the homologous recombination-mediated processes of recombinational DNA repair, DNA transformation, and pilin antigenic variation (reviewed in reference 20). One potential participant in these recombination processes is the Rep helicase. Escherichia coli rep mutants were originally identified by their inability to support phage replication (4, 7). Rep has also been implicated in chromosomal replication (6,24,25), DNA repair, and homologous recombination (4,7,8) A direct link between replication and recombination has been demonstrated for E. coli (21, 36) in a process called replication restart. During this process, replication can be resumed from recombination intermediates formed at stalled replication forks. The E. coli replication restart pathway targets branched DNA structures at the stalled replication fork either by a PriA-dependent pathway that utilizes PriB or PriC and DnaT or by a Rep-dependent pathway that uses PriC and possibly DnaT (35). N. gonorrhoeae does not possess homologues of PriC or DnaT (20), indicating that the secondary pathway of replication restart may be missing from gonococci.Construction of an N. gonorrhoeae rep mutant. In order to study the relationship between replication and recombination in N. gonorrhoeae, we identified the gonococcal rep homologue for further study. N. gonorrhoeae possesses a number of helicases that share homology with E. coli Rep helicase, including Rep, UvrD, and RecB. BLAST analyses performed against the N. gonorrhoeae strain FA1090 genome database (34) revealed that gonococcal Rep has the highest E value (E-134) and shares the greatest sequence identity (48%) and similarity (65%) with E. coli Rep (Fig. 1A). The gonococcal Rep homologue is encoded by a 2,013-bp gene predicted to encode a protein of 671 amino acids. Seven motifs conserved among ATP-dependent DNA helicases (15) are present in Rep (Fig. 1A). Motifs Ia, III, and V are involved in binding single-strand DNA; motifs I and IV are involved in nucleotide binding; and motifs II and VI are important for helicase function (23). Based on the high degree of sequence similarity and the presence of conserved helicase motifs, we conclude that Gc Rep is the homologue of E. coli Rep.Gonococcal rep was cloned and inactivated by inserting a gene encoding tetracycline resistance between codons 303 and 304 (Fig. 1B). To ensure that all phenotypes observed in the rep mutant were due to the rep mutation, complementation analysis was performed with a functional copy of Gc rep elsewhere on the gonococcal chromo...

Mutation of the priA Gene of Neisseria gonorrhoeae Affects DNA Transformation and DNA Repair

“…Overall, the phenotypes associated with a gonococcal priA mutant are consistent with the biological functions of a PriAdependent replication restart pathway demonstrated in E. coli. In contrast, we have shown that the alternative replication restart pathway that is dependent on Rep and PriC does not function in N. gonorrhoeae (18,19). The differences in the viability phenotype and in the repertoire of factors that mediate replication restart in N. gonorrhoeae indicate that evolutionary pressures on the human-specific pathogen N. gonorrhoeae differ significantly from other microbes which occupy multiple environmental niches, and this pressure modifies the basic process of replication in this organism.…”

“…The E. coli replication restart mechanisms require reloading of the DnaB helicase by DnaC, which is targeted to branched structures at the stalled replication fork either by a PriA-dependent pathway that utilizes PriB or PriC and DnaT or by a PriA-independent pathway that uses Rep, PriC, and possibly DnaT (45). N. gonorrhoeae differs from E. coli in that N. gonorrhoeae does not possess homologues of PriC or DnaT (18) and does not have a Rep-dependent replication restart pathway (19), indicating that the overall genetic and biochemical pathway mediating replication restart must be different in the gonococcus than in other bacteria. Furthermore, although PriB and PriC are thought to be redundant in E. coli, the cell requires one of these proteins to be present since an E. coli double mutant is not viable (45,47).…”

“…Numerous gene products are involved in DNA uptake and competence, including pilE, pilQ, and pilT, involved in elaboration of the type IV pilus, and the competence-associated factors comE, comA, comP, comL, tpc, and dca (5). Once transforming homologous DNA is taken up into the cell, it is recombined into the chromosome, a process which requires recA (21) and relies partially on the recBCD-encoded enzyme (33), recN (57), and rep (19) but is independent of the recF-like pathway of recombination (33,57).…”

“…During pilin antigenic variation, a variable portion of a pilS copy replaces the corresponding portion of pilE, recombining at short regions of identity shared between the silent and expressed genes (16). Pilin antigenic variation is dependent on recA (21), the recF-like pathway (33,57), rdgC (32), and rep (19) as well as the ruvABC and recG branch migration pathways (50).…”

Neisseria gonorrhoeae RecQ Helicase HRDC Domains Are Essential for Efficient Binding and Unwinding of the pilE Guanine Quartet Structure Required for Pilin Antigenic Variation

“…N. gonorrhoeae also encodes UvrD and Rep helicases, which share a domain with DNA2 helicase. UvrD does not function during pilin Av (46); however, Rep does have a role (47). Further investigation will determine whether Rep helicase unwinds the pilE G4 structure.…”