Journal article
DNA methylases for site-selective inhibition of type IIS restriction enzyme activity
- Abstract:
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DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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- Files:
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(Preview, Version of record, pdf, 1.6MB, Terms of use)
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- Publisher copy:
- 10.1007/s00253-024-13015-7
Authors
- Publisher:
- Springer
- Journal:
- Applied Microbiology and Biotechnology More from this journal
- Volume:
- 108
- Issue:
- 1
- Article number:
- 174
- Publication date:
- 2024-01-25
- Acceptance date:
- 2024-01-14
- DOI:
- EISSN:
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1432-0614
- ISSN:
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0175-7598
- Language:
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English
- Keywords:
- Pubs id:
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1606502
- Local pid:
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pubs:1606502
- Deposit date:
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2024-01-25
Terms of use
- Copyright holder:
- Flores-Fernández et al
- Copyright date:
- 2024
- Rights statement:
- Copyright © 2024, The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
- Licence:
- CC Attribution (CC BY)
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