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

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01960

DOI=10.3389/fmicb.2020.01960

ISSN=1664-302X

ABSTRACT=<p><underline>M</underline>odification <underline>d</underline>ependent <underline>r</underline>estriction <underline>e</underline>ndonucleases (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<sup>2+</sup> buffer, and promiscuous with Mn<sup>2+</sup>. Ksp11411I is more PT specific with Ni<sup>2+</sup>. 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<sup>+</sup> GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs).</p>