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Updated: Jun 5, 2026

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
Published on: February 7, 2021
Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA
Guang Liu1, Hong-Yu Ou, Tao Wang
1Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Abstract:
Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.
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