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基于蛋白质结构域的序列特异性和硫代磷酸酯依赖性限制性内切核酸酶筛选

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases.

作者信息

Lutz Thomas, Czapinska Honorata, Fomenkov Alexey, Potapov Vladimir, Heiter Daniel F, Cao Bo, Dedon Peter, Bochtler Matthias, Xu Shuang-Yong

机构信息

New England Biolabs, Inc., Ipswich, MA, United States.

International Institute of Molecular and Cell Biology, Warsaw, Poland.

出版信息

Front Microbiol. 2020 Aug 18;11:1960. doi: 10.3389/fmicb.2020.01960. eCollection 2020.

DOI:10.3389/fmicb.2020.01960
PMID:33013736
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7461809/
Abstract

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 Mg buffer, and promiscuous with Mn. Ksp11411I is more PT specific with Ni. 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).

摘要

修饰依赖性限制内切酶（MDREs）可切割修饰的DNA，其序列特异性通常有限（约2 - 4个碱基对）。在此，我们聚焦于那些与HNH内切酶结构域融合了SRA和/或硫结合结构域（SBD）的MDREs，它们可切割胞嘧啶修饰或硫代磷酸化（PT）的DNA。我们独立鉴定了SBD - SRA - HNH内切酶ScoMcrA，它优先切割5 - 羟甲基胞嘧啶（5hmC）修饰的DNA。我们报告了5种SBD - HNH内切酶，它们均识别GpsAAC/GpsTTC序列，并在外侧以单个核苷酸3' 交错切割：EcoWI（N7/N6）、Ksp11411I（N5/N4）、Bsp305I（N6/N4 - 5）、Mae9806I [N(8 - 10)/N(8 - 9)]和Sau43800I [N(8 - 9)/N(7 - 8)]。EcoWI和Bsp305I在Mg缓冲液中对PT修饰的DNA更具特异性，在Mn存在时则较为混杂。Ksp11411I在Ni存在时对PT更具特异性。EcoWI和Ksp11411I可切割完全和半PT修饰的寡核苷酸，而Bsp305I仅切割完全修饰的寡核苷酸。EcoWI在溶液中形成二聚体，在存在两个修饰位点时切割效率更高。此外，我们证明EcoWI的PT依赖性活性具有生物学功能：表达EcoWI的细胞对dnd GpsAAC修饰的质粒有强烈限制作用，对GpsGCC DNA的限制作用较弱。这项工作为PT依赖性限制内切酶（PTDRs）的生物技术应用建立了一个框架。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/db5bf9ffb710/fmicb-11-01960-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/c5514b26432f/fmicb-11-01960-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/1b0099b6cdb8/fmicb-11-01960-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/e31f91dce4ef/fmicb-11-01960-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/78cf76ab60ec/fmicb-11-01960-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/9cceaad0b7a0/fmicb-11-01960-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/db5bf9ffb710/fmicb-11-01960-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/c5514b26432f/fmicb-11-01960-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/1b0099b6cdb8/fmicb-11-01960-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/e31f91dce4ef/fmicb-11-01960-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/78cf76ab60ec/fmicb-11-01960-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/9cceaad0b7a0/fmicb-11-01960-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d034/7461809/db5bf9ffb710/fmicb-11-01960-g006.jpg

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SspABCD-SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities.SspABCD-SspE 是一种硫代磷酸化感应细菌防御系统，具有广泛的抗噬菌体活性。
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A protein architecture guided screen for modification dependent restriction endonucleases.
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PLoS Genet. 2022 Sep 19;18(9):e1010389. doi: 10.1371/journal.pgen.1010389. eCollection 2022 Sep.
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A new type of DNA phosphorothioation-based antiviral system in archaea.古菌中一种新型基于 DNA 硫代磷酸化的抗病毒系统。
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