Pseudomonas putida MPE, a manganese-dependent endonuclease of the binuclear metallophosphoesterase superfamily, incises single-strand DNA in two orientations to yield a mixture of 3'-PO4 and 3'-OH termini.
Nucleic Acids Res
; 49(2): 1023-1032, 2021 01 25.
Article
en En
| MEDLINE
| ID: mdl-33367848
ABSTRACT
Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3' and 5' single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3'-OH and 3'-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
ADN de Cadena Simple
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Pseudomonas putida
/
Enzimas Reparadoras del ADN
/
Desoxirribonucleasa I
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos