A single active site in the mariner transposase cleaves DNA strands of opposite polarity
The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste trans...
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| Format: | Article |
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Oxford University Press
2017
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| Online Access: | https://eprints.nottingham.ac.uk/47838/ |
| _version_ | 1848797642524459008 |
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| author | Claeys Bouuaert, Corentin Chalmers, Ronald |
| author_facet | Claeys Bouuaert, Corentin Chalmers, Ronald |
| author_sort | Claeys Bouuaert, Corentin |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease. |
| first_indexed | 2025-11-14T20:07:07Z |
| format | Article |
| id | nottingham-47838 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:07:07Z |
| publishDate | 2017 |
| publisher | Oxford University Press |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-478382020-05-04T19:07:18Z https://eprints.nottingham.ac.uk/47838/ A single active site in the mariner transposase cleaves DNA strands of opposite polarity Claeys Bouuaert, Corentin Chalmers, Ronald The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease. Oxford University Press 2017-09-19 Article PeerReviewed Claeys Bouuaert, Corentin and Chalmers, Ronald (2017) A single active site in the mariner transposase cleaves DNA strands of opposite polarity. Nucleic Acids Research, gkx826 . ISSN 1362-4962 https://doi.org/10.1093/nar/gkx826 doi:10.1093/nar/gkx826 doi:10.1093/nar/gkx826 |
| spellingShingle | Claeys Bouuaert, Corentin Chalmers, Ronald A single active site in the mariner transposase cleaves DNA strands of opposite polarity |
| title | A single active site in the mariner transposase cleaves DNA strands of opposite polarity |
| title_full | A single active site in the mariner transposase cleaves DNA strands of opposite polarity |
| title_fullStr | A single active site in the mariner transposase cleaves DNA strands of opposite polarity |
| title_full_unstemmed | A single active site in the mariner transposase cleaves DNA strands of opposite polarity |
| title_short | A single active site in the mariner transposase cleaves DNA strands of opposite polarity |
| title_sort | single active site in the mariner transposase cleaves dna strands of opposite polarity |
| url | https://eprints.nottingham.ac.uk/47838/ https://eprints.nottingham.ac.uk/47838/ https://eprints.nottingham.ac.uk/47838/ |