TY - JOUR

T1 - Cloning and characterization of the major AP endonuclease from Staphylococcus aureus

AU - Turgimbayeva, Aigerim

AU - Zein, Ulan

AU - Zharkov, Dmitry O.

AU - Ramankulov, Yerlan

AU - Saparbaev, Murat

AU - Abeldenov, Sailau

N1 - Funding Information: This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP08856811 ) to S.A. D.O.Z. acknowledges support from Russian Foundation for Basic Research ( 20-04-00554-а ) and partial salary support from Russian Ministry of Science and Education (AAAA-A20–120101690009-7 ). M.S. was supported by Electricité de France ( RB 2021-05 ) (http://www.edf.fr) and Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP09260233 ). Publisher Copyright: © 2022 Elsevier B.V.

PY - 2022/11

Y1 - 2022/11

N2 - 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 β-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.

AB - 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 β-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.

KW - Abasic sites

KW - AP endonuclease

KW - Base excision repair

KW - DNA damage

KW - DNA repair

KW - Endonuclease IV

KW - Nucleotide incision repair

KW - Oxidative damage

KW - Staphylococcus aureus

KW - DNA/metabolism

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism

KW - Hydrogen Peroxide

KW - Deoxyribonuclease IV (Phage T4-Induced)/chemistry

KW - Nucleotides

KW - Escherichia coli/metabolism

KW - DNA Repair

KW - Cloning, Molecular

KW - Staphylococcus aureus/genetics

KW - DNA Damage

KW - Endonucleases/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85137352207&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/dc268cd4-68cd-35c2-b7e8-92feab4ab38c/

U2 - 10.1016/j.dnarep.2022.103390

DO - 10.1016/j.dnarep.2022.103390

M3 - Article

C2 - 36088709

AN - SCOPUS:85137352207

VL - 119

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

M1 - 103390

ER -