Abstract
Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV’s function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0–9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.
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Acknowledgements
We thank Prof. Li Huang at Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, for kindly providing the genomic DNA of the Sulfolobus islandicus REY15A.
Funding
This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20191219), High Level Talent Support Program of Yangzhou University and the Academic Leader of Middle and Young People of Yangzhou University Grant to LZ, Jiangsu Province Practice Innovation Training Program for College Students (202211117084Y) to JS, and Open-end Funds of Jiangsu Key Laboratory of Marine Bioresources and Environment (SH20211206) to QL.
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LZ, YG, XL and PO: wrote the main manuscript text. LZ: prepared figure 1. YY, JS and QL: prepared figures 2-5 and 7-8. YG: prepared figure 6. YY, LZ, JS and QL: prepared all supplmental figures. All authors reviewed the manuscript.
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Yin, Y., Shi, J., Zhang, L. et al. Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A. World J Microbiol Biotechnol 39, 90 (2023). https://doi.org/10.1007/s11274-023-03526-2
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DOI: https://doi.org/10.1007/s11274-023-03526-2