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DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island
Relaxases of the MOB(H) family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 d...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736028/ https://www.ncbi.nlm.nih.gov/pubmed/31276596 http://dx.doi.org/10.1093/nar/gkz577 |
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author | Heilers, Jan-Hendrik Reiners, Jens Heller, Eva-Maria Golzer, Annika Smits, Sander H J van der Does, Chris |
author_facet | Heilers, Jan-Hendrik Reiners, Jens Heller, Eva-Maria Golzer, Annika Smits, Sander H J van der Does, Chris |
author_sort | Heilers, Jan-Hendrik |
collection | PubMed |
description | Relaxases of the MOB(H) family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOB(H) family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn(2+) or Co(2+) dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3′ of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site. |
format | Online Article Text |
id | pubmed-6736028 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-67360282019-09-16 DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island Heilers, Jan-Hendrik Reiners, Jens Heller, Eva-Maria Golzer, Annika Smits, Sander H J van der Does, Chris Nucleic Acids Res Nucleic Acid Enzymes Relaxases of the MOB(H) family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOB(H) family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn(2+) or Co(2+) dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3′ of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site. Oxford University Press 2019-09-05 2019-07-05 /pmc/articles/PMC6736028/ /pubmed/31276596 http://dx.doi.org/10.1093/nar/gkz577 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Nucleic Acid Enzymes Heilers, Jan-Hendrik Reiners, Jens Heller, Eva-Maria Golzer, Annika Smits, Sander H J van der Does, Chris DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island |
title | DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island |
title_full | DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island |
title_fullStr | DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island |
title_full_unstemmed | DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island |
title_short | DNA processing by the MOB(H) family relaxase TraI encoded within the gonococcal genetic island |
title_sort | dna processing by the mob(h) family relaxase trai encoded within the gonococcal genetic island |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736028/ https://www.ncbi.nlm.nih.gov/pubmed/31276596 http://dx.doi.org/10.1093/nar/gkz577 |
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