Purification and characterization of the RecA protein from Neisseria gonorrhoeae

The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA_Ng), we overexpressed and purified the RecA_Ng and SSB_Ng proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA_Ng promoted more strand exchange at early time points than RecA_Ec through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA_Ng is more efficient at displacing SSB from ssDNA and that RecA_Ng shows higher ATPase activity during strand exchange than RecA_Ec. Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA_Ec catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA_Ng catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA_Ng than in RecA_Ec. This difference may explain the unusually high ATPase activity observed for RecA_Ng with the strand exchange activity between RecA_Ng and RecA_Ec being more similar.