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'-PO(4) and 3'-OH termini Journal Article
| Authors: | Ghosh, S.; Ejaz, A.; Repeta, L.; Shuman, S. |
| Article Title: | 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'-PO(4) and 3'-OH termini |
| 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. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. |
| Journal Title: | Nucleic Acids Research |
| Volume: | 49 |
| Issue: | 2 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2021-01-25 |
| Start Page: | 1023 |
| End Page: | 1032 |
| Language: | English |
| DOI: | 10.1093/nar/gkaa1214 |
| PUBMED: | 33367848 |
| PROVIDER: | scopus |
| PMCID: | PMC7826289 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 1 March 2021 -- Source: Scopus |
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