Characterization of RecA and LexA, key regulatory proteins of the Staphylococcus aureus SOS response

Soon after the introduction of penicillin antibiotic in 1940s, penicillin-resistant strains including resistant S. aureus were identified. This led to the discovery of many other antibiotics, which consequently propagated in acquiring multidrug resistance in bacteria. Currently, the control of S. aureus has become a major concern and a potent drug is yet to be discovered. The resistance towards the antibiotics is easily acquired through chromosomal mutations and acquisition of horizontally transferred genes among bacterial and other host species. Both processes are directly controlled by the central recombinase RecA protein in bacteria. Antibiotics are known to cause DNA damage, directly or indirectly inducing bacterial SOS response, which is responsible for higher rates of induced-mutagenesis in many bacteria. A key initial molecular event of SOS repose is the interaction of RecA, the positive regulator, and LexA, the only known SOS repressor. As published, E. coli and S. aureus cannot acquire antibiotic resistance significantly under SOS response deficient conditions such as in the presence of noncleavable LexA [1, 2]. In this study, we report the initial in vitro characterization of these key regulatory proteins, RecA and LexA, and their interaction. The findings could be used in producing antistaphylococcal agents.;S. aureus RecA, LexA and two SSB proteins (Sa SSB1 and Sa SSB2) were overexpressed in E. coli cells and purified to near homogeneity. The proteins were subjected to RecA-promoted LexA cleavage in an assay. According to the data obtained, Sa RecA does mediate the Sa LexA cleavage; albeit in a slower process compared to that in E. coli. The reaction occurs more efficiently in the presence of the non-hydrolyzable ATP analogue, ATPgammaS, than hydrolysable ATP. RecA bound to DNA hydrolyzes ATP and also detaches from DNA upon hydrolysis. A stable RecA nucleoprotein filament is required for LexA to cleave under the given conditions. Hence, the data suggests possible defects in the formation of a stabilized Sa RecA nucleoprotein filament in the presence of the natural cofactor ATP.;An active RecA nucleoprotein filament is also required to mediate the main RecA function, the homologous recombination. We then evaluated the recombinase activity using three-strand DNA exchange and D-loop assays. Formation of product is observed in D-loop assay but not in the classical exchange assay. The resulted D-loop product is an indication of Sa RecA recombinase activity. ATPase activity of Sa RecA was evaluated as it was shown to be required for branch migration of long DNA substrates during strand exchange. The data showed that Sa RecA is an ATPase. However, the addition of SSB protein that is used to melt secondary DNA structures in ATPase assay indicated a displacement of Sa RecA from DNA. It appeared that other accessory proteins or factors may be required for facilitating Sa RecA functions. The assays done in the presence of Ec DinI, an Ec RecA filament stabilizer, together with SSB, significantly enhanced the Sa RecA-mediated DNA strand exchange as well as LexA autocleavage. Further, Ec-DinI stabilized Sa RecA nucleoprotein filaments compete better with any of the SSB proteins (Ec SSB, Sa SSB1, or Sa SSB2) added. Another naturally available cofactor dATP that was shown to bind RecA tightly to DNA also could stimulate these activities even in the absence of Ec DinI.;Taken together, the work in this dissertation concludes that Sa RecA is highly dependent on nucleofilament stabilizing accessory proteins or other factors to mediate its functions efficiently. By providing that, Sa RecA functions as a recombinase and a coprotease. Hence, it is likely that the hypothesized key molecular interactions between Sa RecA and Sa LexA in vivo are similar to E. coli SOS response. This work would provide the initiative in producing drugs to hinder this interaction, thereby minimizing S. aureus acquiring antibiotic resistance.