Abstract
A recently identified and widely prevalent prokaryal gene cluster encodes a suite of enzymes with imputed roles in nucleic acid repair. The enzymes are as follows: MPE, a DNA endonuclease; Lhr-Core, a 3'-5' DNA helicase; LIG, an ATP-dependent DNA ligase; and Exo, a metallo-β-lactamase-family nuclease. Bacterial and archaeal MPE proteins belong to the binuclear metallophosphoesterase superfamily that includes the well-studied DNA repair nucleases Mre11 and SbcD. Here, we report that the Pseudomonas putida MPE protein is a manganese-dependent DNA endonuclease that incises either linear single strands or the single-strand loops of stem-loop DNA structures. MPE has feeble activity on duplex DNA. A crystal structure of MPE at 2.2 Å resolution revealed that the active site includes two octahedrally coordinated manganese ions. Seven signature amino acids of the binuclear metallophosphoesterase superfamily serve as the enzymic metal ligands in MPE: Asp33, His35, Asp78, Asn112, His124, His146, and His158 A swath of positive surface potential on either side of the active site pocket suggests a binding site for the single-strand DNA substrate. The structure of MPE differs from Mre11 and SbcD in several key respects: (i) MPE is a monomer, whereas Mre11 and SbcD are homodimers; (ii) MPE lacks the capping domain present in Mre11 and SbcD; and (iii) the topology of the β sandwich that comprises the core of the metallophosphoesterase fold differs in MPE vis-à-vis Mre11 and SbcD. We surmise that MPE exemplifies a novel clade of DNA endonuclease within the binuclear metallophosphoesterase superfamily.
Highlights
A recently identified and widely prevalent prokaryal gene cluster encodes a suite of enzymes with imputed roles in nucleic acid repair
We reported that P. putida MPE is a manganese-dependent phosphodiesterase that released p-nitrophenol from bis-p-nitrophenyl phosphate and p-nitrophenyl-5Ј-thymidylate but displayed no detectable phosphomonoesterase activity against p-nitrophenyl phosphate
We examine in greater detail the nuclease activity of P. putida MPE and find it to be a manganese-dependent single-strand DNA endonuclease that incises either linear single strands or the single-stranded loops of DNA stem-loop structures
Summary
Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021. Tel.: 212-639-7145; E-mail: s-shuman@ ski.mskcc.org. MPE is a manganese-dependent DNA nuclease that sequentially converted a closed circle plasmid DNA to nicked circle and linear forms prior to converting the linear DNA to smaller fragments [1]. This initial characterization of MPE suggested that it might be a new bacterial homolog of the archaeal and eukaryal DNA nuclease Mre and the homologous bacterial DNA nuclease SbcD, which are binuclear metallophosphoesterases [5,6,7,8,9,10,11,12]. We discuss how the structure of MPE differs significantly from Mre and SbcD
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