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.

摘要: Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. is encoded widely conserved repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for nuclease activity, albeit inessential hydrolysis bis-p-nitrophenylphosphate. We propose that contacts scissile phosphodiester serves general acid catalyst to expel OH leaving group product strand. find cleaves 3' 5' single-strands tailed duplex DNAs can sense incise duplexes at sites short mismatch bulges opposite nick. show an ambidextrous phosphodiesterase capable hydrolyzing ssDNA backbone in either orientation generate mixture 3'-OH 3'-PO4 cleavage products. The directionality dictated by water nucleophile vis-a-vis group, which must be near apical reaction proceed. active site metal-bound are invariant enzyme bind productively orientations.