Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases
Name
fmicb-11-01960.pdf
Description
Published version
Size
2.93 MB
Format
Unknown
Checksum (MD5)
d82310c31589c5540efea85f3953706a
Author(s)
Lutz, Thomas
Czapinska, Honorata
Fomenkov, Alexey
Potapov, Vladimir
Heiter, Daniel F
Cao, Bo
Dedon, Peter
Bochtler, Matthias
Xu, Shuang-yong
Date Issued
August 2020
Journal
Frontiers in Microbiology
Publisher
Frontiers Media SA
Version
Final published version
Abstract
© 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).
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Creative Commons Attribution 4.0 International license
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DOI of Published Version
https://doi.org/10.3389/FMICB.2020.01960
