title: Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.
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.
authors.affiliation: Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Liu
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Ou
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Huarong
authors.initials: H
authors.lastname: Tan
authors.forename: Xiufen
authors.initials: X
authors.lastname: Zhou
authors.forename: Kumar
authors.initials: K
authors.lastname: Rajakumar
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.forename: Xinyi
authors.initials: X
authors.lastname: He
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics; D048170:Streptomyces lividans / Q000235:genetics / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010710:Phosphates; D013455:Sulfur; D004262:DNA Restriction Enzymes; C035638:thiophosphoric acid