Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A.
World J Microbiol Biotechnol
; 39(4): 90, 2023 Feb 08.
Article
em En
| MEDLINE
| ID: mdl-36752840
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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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sulfolobus
/
Desoxirribonuclease (Dímero de Pirimidina)
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
World J Microbiol Biotechnol
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
China
País de publicação:
ALEMANHA
/
ALEMANIA
/
DE
/
DEUSTCHLAND
/
GERMANY