Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases
© Copyright © 2020 Lutz, Czapinska, Fomenkov, Potapov, Heiter, Cao, Dedon, Bochtler and Xu. Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2–4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domai...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media SA
2021
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| Online Access: | https://hdl.handle.net/1721.1/133512 |
| _version_ | 1826214103928012800 |
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| author | Lutz, Thomas Czapinska, Honorata Fomenkov, Alexey Potapov, Vladimir Heiter, Daniel F Cao, Bo Dedon, Peter Bochtler, Matthias Xu, Shuang-yong |
| author_facet | Lutz, Thomas Czapinska, Honorata Fomenkov, Alexey Potapov, Vladimir Heiter, Daniel F Cao, Bo Dedon, Peter Bochtler, Matthias Xu, Shuang-yong |
| author_sort | Lutz, Thomas |
| collection | MIT |
| description | © Copyright © 2020 Lutz, Czapinska, Fomenkov, Potapov, Heiter, Cao, Dedon, Bochtler and Xu. Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2–4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3′ stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I [N(8-10)/N(8-9)], and Sau43800I [N(8-9)/N(7-8)]. EcoWI and Bsp305I are more specific for PT modified DNA in Mg2+ buffer, and promiscuous with Mn2+. Ksp11411I is more PT specific with Ni2+. EcoWI and Ksp11411I cleave fully- and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependent activity has biological function: EcoWI expressing cells restrict dnd+ GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs). |
| first_indexed | 2024-09-23T15:59:33Z |
| format | Article |
| id | mit-1721.1/133512 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T15:59:33Z |
| publishDate | 2021 |
| publisher | Frontiers Media SA |
| record_format | dspace |
| spelling | mit-1721.1/1335122021-10-28T03:15:19Z Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases Lutz, Thomas Czapinska, Honorata Fomenkov, Alexey Potapov, Vladimir Heiter, Daniel F Cao, Bo Dedon, Peter Bochtler, Matthias Xu, Shuang-yong © Copyright © 2020 Lutz, Czapinska, Fomenkov, Potapov, Heiter, Cao, Dedon, Bochtler and Xu. Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2–4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3′ stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I [N(8-10)/N(8-9)], and Sau43800I [N(8-9)/N(7-8)]. EcoWI and Bsp305I are more specific for PT modified DNA in Mg2+ buffer, and promiscuous with Mn2+. Ksp11411I is more PT specific with Ni2+. EcoWI and Ksp11411I cleave fully- and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependent activity has biological function: EcoWI expressing cells restrict dnd+ GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs). 2021-10-27T19:53:16Z 2021-10-27T19:53:16Z 2020 2021-08-26T15:33:19Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/133512 en 10.3389/FMICB.2020.01960 Frontiers in Microbiology Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf Frontiers Media SA Frontiers |
| spellingShingle | Lutz, Thomas Czapinska, Honorata Fomenkov, Alexey Potapov, Vladimir Heiter, Daniel F Cao, Bo Dedon, Peter Bochtler, Matthias Xu, Shuang-yong Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases |
| title | Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases |
| title_full | Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases |
| title_fullStr | Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases |
| title_full_unstemmed | Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases |
| title_short | Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases |
| title_sort | protein domain guided screen for sequence specific and phosphorothioate dependent restriction endonucleases |
| url | https://hdl.handle.net/1721.1/133512 |
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