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Mol Microbiol. 2018 Nov;110(3):484-497. doi: 10.1111/mmi.14118. Epub 2018 Oct 5.

Identification of a conserved DNA sulfur recognition domain by characterizing the phosphorothioate-specific endonuclease SprMcrA from Streptomyces pristinaespiralis.

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1
State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

Abstract

Streptomyces species have been valuable models for understanding the phenomenon of DNA phosphorothioation in which sulfur replaces a non-bridging oxygen in the phosphate backbone of DNA. We previously reported that the restriction endonuclease ScoMcrA from Streptomyces coelicolor cleaves phosphorothioate DNA and Dcm-methylated DNA at sites 16-28 nucleotides away from the modification sites. However, cleavage of modified DNA by ScoMcrA is always incomplete and accompanied by severe promiscuous activity on unmodified DNA. These features complicate the studies of recognition and cleavage of phosphorothioate DNA. For these reasons, we here characterized SprMcrA from Streptomyces pristinaespiralis, a much smaller homolog of ScoMcrA with a rare HRH motif, a variant of the HNH motif that forms the catalytic center of these endonucleases. The sulfur-binding domain of SprMcrA and its phosphorothioation recognition site were determined. Compared to ScoMcrA, SprMcrA has higher specificity in discerning phosphorothioate DNA from unmodified DNA, and this enzyme generally cuts both strands at a distance of 11-14 nucleotides from the 5' side of the recognition site. The HRH/HNH motif has its own sequence specificity in DNA hydrolysis, leading to failure of cleavage at some phosphorothioated sites. An R248N mutation of the central residue in HRH resulted in 30-fold enhancement in cleavage activity of phosphorothioate DNA and altered the cleavage efficiency at some sites, whereas mutation of both His residues abolished restriction activity. This is the first report of a recognition domain for phosphorothioate DNA and phosphorothioate-dependent and sequence-specific restriction activity.

PMID:
30184284
DOI:
10.1111/mmi.14118

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