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

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.

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

“…The endonuclease activity of the two proteins was assayed in the presence of various divalent metal cations. Both were active in Mn 2+ buffer and partially active in Co 2+ buffer as previously reported (data not shown) (Yu et al, 2018). SprMcrA-S ( 87 aa, N-terminal deletion variant) displayed partial endonuclease activity on pBR322 and dnd + plasmids in Mn 2+ buffer.…”

“…We conclude from the above results that the N-terminal 87 aa form part of the SprMcrA domain that recognizes PT-modified DNA. Similar observation was first demonstrated in a previous work (Yu et al, 2018). Removal of this N-terminal domain converts the enzyme to a non-specific nicking endonuclease (NEase).…”

“…It has been reported that WT SprMcrA carries the H-N-R catalytic residues in the HNH nuclease domain. Substitution of the Arg residue by Asn resulted in ∼40-fold increase of the enzyme activity (Yu et al, 2018).…”

“…The domain organization of SprMcrA and SprMcrA-S is shown in Figure 2A. SprMcrA was shown to specifically cleave phosphorothioated DNA, at wobbled distance from the site of modification (Yu et al, 2018). SprMcrA and SprMcrA-S were overexpressed in E. coli as intein-CBD fusions and purified by chitin affinity chromatography.…”

“…Removal of this N-terminal domain converts the enzyme to a non-specific nicking endonuclease (NEase). The original SprMcrA paper reported that some downstream sequences were nicked by the enzyme near the PT-modified sites (Yu et al, 2018), suggesting that SprMcrA may have both dsDNA cleavage and nicking activity. The sequence specificity of the HNH domain of SprMcrA may partially explain why some of the modified sites in pUC19, phosphorothioated by passing through dnd + E. coli host, were not cleaved efficiently (Yu et al, 2018).…”