Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases
Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases
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DOI：
10.3389/fmicb.2020.01960
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发表时间：
2020-08-18
影响因子：
5.2
通讯作者：
Xu, Shuang-yong
中科院分区：
文献类型：
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作者：
Lutz, Thomas;Czapinska, Honorata;Xu, Shuang-yong
修饰依赖的限制性内切酶(MDRE)限制修饰的DNA，通常具有有限的序列特异性(类似于2-4bp)。在这里，我们集中在MDRE具有SRA和/或SBD(硫磺结合结构域)融合到HNH内切酶结构域，切割胞嘧啶修饰或硫代(PT)DNA。我们独立鉴定了SBD-SRA-HNH内切酶ScoMcrA，它优先切割5hmC修饰的DNA。我们报道了5种SBD-HNH内切酶，它们都能识别GpsAAC/GpsTTC序列，并用单个核苷酸3‘错开切割：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对Mg(2+)缓冲溶液中PT修饰的DNA有较高的特异性，与Mn2+有混杂作用。Ksp11411I对Ni2+具有更强的PT特异性。EcoWI和Ksp11411I切割完全和半PT修饰的寡核苷酸，而Bsp305I只切割完全修饰的寡核苷酸。EcoWI在溶液中形成二聚体，并且在存在两个修饰位点时更有效地切割。此外，我们还证明了EcoWIPT依赖的活性具有生物学功能：表达EcoWI的细胞强烈地限制了DND(+)GpsAAC修饰的质粒，而GpsGCC DNA则弱限制了DND(+)GpsAAC的修饰。这项工作为PT依赖的限制性内切酶(PTDR)的生物技术应用建立了一个框架。
Modificationdependentrestrictionendonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (similar to 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(2+)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).