Biochemical Assessment of the Mutant Sliding ß-Clamp on Stimulation of Endonuclease IV from Staphylococcus aureus.

Staphylococcus aureus is a pathogenic bacterium that causes various infections in humans. The emergence of methicillin-resistant Staphylococcus aureus makes treatment more challenging. Recent research has shown that bacterial ß-clamp is not only a processivity factor but can also stimulate the activity of other enzymes of DNA metabolism. This article examines the interaction between apurinic/apyrimidinic (AP) endonuclease IV (Nfo) and ß-clamp from Staphylococcus aureus, which has not been previously researched. Recombinant DNA repair enzymes, beta-clamp, were cloned, expressed, and purified. Biochemical methods were employed to assess the stimulation of beta-clamp-activated AP endonuclease activity of Nfo. We demonstrated that mutations in the C-terminal conserved region led to disruption of stimulation of Nfo AP endonuclease activity. The study provides evidence of a specific interaction between Nfo and ß-clamp, which suggests that ß-clamp may play a more direct role in DNA repair processes than previously thought. These findings have important implications for understanding the mechanism of DNA repair, particularly in relation to the role of ß-clamp. Understanding the underlying mechanisms of interaction between DNA metabolism enzymes can aid in predicting new drug targets for antibiotic resistance battle. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01148-8.

Cloning and characterization of the major AP endonuclease from Staphylococcus aureus.

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Complete genome analysis of Staphylococcus aureus identified a single AP endonuclease, SaNfo, which is a member of the endonuclease IV family exemplified by Escherichia coli Nfo. At present, it remains unknown whether SaNfo possesses DNA repair activities similar to its counterparts from E. coli and other bacteria. Here, we report that the purified SaNfo protein contains efficient AP endonuclease and nucleotide incision repair (NIR) activities. Optimal reaction conditions for SaNfo-catalysed AP endonuclease activity are high ionic strength and Mn2+ concentration, pH in range 7.5-9.0 and the temperature optimum of 37-45 °C. Cell-free extracts of S. aureus exhibited efficient AP site cleavage and NIR activities. Heterologous expression of SaNfo strongly reduces the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2. Site-directed mutagenesis showed that the Glu258 residue is critical for the SaNfo enzyme function. The AP endonuclease but not the NIR activity of SaNfo were stimulated by the ß-clamp (SaDnaN dimer), suggesting that it might participate in the organization of BER in S. aureus. Overall, our data confirm that the activity, substrate specificity and in vivo functionality of S. aureus Nfo are consistent with this protein being the major AP endonuclease for the repair of DNA damage generated by endogenous and host-imposed factors.