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 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).
Highlights
DNA backbone phosphorothioation (PT or ps) is an alternative to DNA base methylation for distinguishing host from invading DNA in bacteria
The identical dinucleotides from three original bacterial genomes were detected
In this work we independently confirmed that the enzyme has 5hmC-stimulated activity on modified PCR DNA in vitro and modified T4gt phage DNA in vivo
Summary
DNA backbone phosphorothioation (PT or ps) is an alternative to DNA base methylation for distinguishing host from invading DNA in bacteria. Modified DNA has sulfur atoms in the pro-R (or Rp) conformation (Zhou et al, 2005). Sequence-dependent PT modifications are incorporated by the DndABCDE (DndA-E) enzyme complex. DndA is often replaced by a cysteine desulfurase such as IscS in E. coli (Liang et al, 2007; Wang et al, 2019). DndB serves as a negative regulator of the dnd operon and its inactivation can significantly increase the level of PT modification. PT modifications are usually introduced sequence .
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