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.

Draft Genome Sequence of Lactobacillus salivarius KZ-NCB, Isolated from Chicken Cecum.

Here, we report the draft genome sequence of Lactobacillus salivarius strain KZ-NCB, which was isolated from the cecum of a healthy chicken from a poultry farm in Kazakhstan.

Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori.

Apurinic/apyrimidinic (AP) endonucleases play critical roles in the repair of abasic sites and strand breaks in DNA. Complete genome sequences of Helicobacter pylori reveal that this bacterial specie has a single AP endonuclease. An H. pylori homolog of Xth (HpXth) is a member of exonuclease III family, which is represented by Escherichia coli Xth. Currently, it remains unknown whether this single AP endonuclease has DNA repair activities similar to those of its counterpart in E. coli and other bacteria. We report that HpXth possesses efficient AP site cleavage, 3'-repair phosphodiesterase, and 3'-phosphatase activities but not the nucleotide incision repair function. Optimal reaction conditions for HpXth's AP endonuclease activity are low ionic strength, high Mg2+ concentration, pH in the range 7-8, and temperature 30 °C. The kinetic parameters measured under steady-state conditions showed that HpXth removes the AP site, 3'-blocking sugar-phosphate, and 3'-terminal phosphate in DNA strand breaks with good efficiency (kcat/KM = 1240, 44, and 5,4 µM-1·min-1, respectively), similar to that of E. coli Xth. As expected, the presence of HpXth protein in AP endonuclease-deficient E. coli xth nfo strain significantly reduced the sensitivity to an alkylating agent and H2O2. Mutation of active site residue D144 in HpXth predicted to be essential for catalysis resulted in a complete loss of enzyme activities. Several important structural features of HpXth were uncovered by homology modeling and phylogenetic analysis. Our data show the DNA substrate specificity of H. pylori AP endonuclease and suggest that HpXth counteracts the genotoxic effects of DNA damage generated by endogenous and host-imposed factors.

Obtaining and characterization of monoclonal antibodies against recombinant extracellular domain of human epidermal growth factor receptor 2.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for detection and treatment of different types of cancers such as breast, ovarian, stomach cancer. In this study, we developed a monoclonal antibody against the extracellular domain (ECD) of HER2 biomarker of breast cancer. For this purpose, the ECD-HER2 gene was amplified and cloned into an expression vector. Gene was generated in Escherichia coli BL21 (DE3) strain for expression of recombinant protein. The expressed protein was separated by SDS-PAGE and detected by anti-his monoclonal antibody in immunoblotting. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells injected with recombinant ECD-HER2 and screened by ELISA for the production of monoclonal antibody. The results indicate that out of three candidate hybridoma cells one clone (1E7) was producing the highest titer and antibody specificity was envisioned in ELISA results. In vivo scaling up culture of hybridoma cells in BALB/C mice lead to significant increase in the monoclonal antibody concentration up to 16 mg/ml. Immunochemical methods demonstrated the specificity of developed antibody against ECD-HER2 protein.