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

Abstract

The strict human pathogen Neisseria gonorrhoeae utilizes homologous recombination to antigenically vary the pilus, thus evading the host immune response. High-frequency gene conversion reactions between many silent pilin loci and the expressed pilin locus (pilE) allow for numerous pilus variants per strain to be produced from a single strain. For pilin antigenic variation (Av) to occur, a guanine quartet (G4) structure must form upstream of pilE. The RecQ helicase is one of several recombination or repair enzymes required for efficient levels of pilin Av, and RecQ family members have been shown to bind to and unwind G4 structures. Additionally, the vast majority of RecQ helicase family members encode one "helicase and RNase D C-terminal" (HRDC) domain, whereas the N. gonorrhoeae RecQ helicase gene encodes three HRDC domains, which are critical for pilin Av. Here, we confirm that deletion of RecQ HRDC domains 2 and 3 causes a decrease in the frequency of pilin Av comparable to that obtained with a functional knockout. We demonstrate that the N. gonorrhoeae RecQ helicase can bind and unwind the pilE G4 structure. Deletion of the RecQ HRDC domains 2 and 3 resulted in a decrease in G4 structure binding and unwinding. These data suggest that the decrease in pilin Av observed in the RecQ HRDC domain 2 and 3 deletion mutant is a result of the enzyme's inability to efficiently bind and unwind the pilE G4 structure.

Initiation of homologous recombination at DNA nicks

“…Antigen variation depends on well-characterized repair factors: RecJ, a 5′-3′ exonuclease; RecA, which promotes strand exchange; and RecF pathway proteins including RecQ, a G4 helicase (23,24). It does not depend on the RecBCD pathway that promotes HDR at DSBs (25).…”

Analyzing Neisseria gonorrhoeae Pilin Antigenic Variation Using 454 Sequencing Technology

“…RecQ family members are also important helicases for dissolving G4 structures (58)(59)(60). The gonococcal RecQ has a unique triplication of the C-terminal HRDC domain that is required for pilin Av (61,62). An insertion in recQ was originally identified using a PCR-based assay (21,63) and a PDCMC screen for pilin Avd mutants (56).…”

Neisseria gonorrhoeae MutS Affects Pilin Antigenic Variation through Mismatch Correction and Not by pilE Guanine Quartet Binding

“…The N. gonorrhoeae RecA and RecQ proteins have already been shown to bind to the pilE G4 in vitro (51,52). RecA is required for pilin AV through promotion of single-strand annealing and DNA strand exchange in homologous recombination, and the pilE G4 may serve to recruit RecA, facilitating strand exchange.…”

“…RecA is required for pilin AV through promotion of single-strand annealing and DNA strand exchange in homologous recombination, and the pilE G4 may serve to recruit RecA, facilitating strand exchange. RecQ is a 3=¡5= helicase that is proposed to unwind secondary structures, and mutations to recQ severely decrease the rate of pilin AV (51,53). Another protein candidate for binding to G4s is MutS, whose eukaryotic MutS␣ homolog activates the immunoglobulin switch recombination through G4 binding (54).…”