Functional Studies On A Type Ⅳ Restriction Enzyme ScoMcrA From Streptomyces Coelicolor M145

As the hereditary material, DNA is composed of adenine, thymine, cytosine, guanine as well as methylated bases, and used to be considered generally constitute of atoms C, H, O, N, P five elements. Most recent studies revealed that sulfur was naturally incorporated as phosphorothioate in some bacterial DNA.As we know, DNA modification is always associated with restriction enzyme in bacteria. In the arm race between bacteria and phages, bacteria developed some modification-dependent restriction enzymes to prevent the entry of alien DNA with different modification pattern from its own.In this study, we observed aberrant expression of DNA phosphorothioation encoding gene cluster dnd in Streptomyces coelicolor in which we determined a phosphorothioation dependent restriction system and its coding gene sco McrA. Interestingly, dnd gene cluster from S. lividans and scoMcr A from S. coelicolor are present in two mutually exclusive mobile genomic islands.In the attempt to purify ScoMcrA, unexpectedly scoMcrA could not be introduced in E. coli strains of dcm genotype. Purified ScoMcrA from BL21(DE3) specifically recognizes the phosphorothioated DNA and makes double strands cleavage at 17 nt ~25 nt away from the sulfur modification site when supplied with manganese ion in vitro. As well, Sco McrA recognizes and cleaves the Dcm methylated DNA. The cleavage sites of ScoMcrA on Dcm methylated DNA were 12 nt ~ 16 nt away from 5-methylated cytosine on flanking sides. This is the first report of an endonuclease that targets two entirely different modifications.The structure of apo-ScoMcrA and the complex of partial ScoMcrA and its substrate phosphorothioated DNA were resolved and revealed the mechanism by which ScoMcr A binds phosphorothioated DNA and Dcm methylated DNA with two separate domains and makes cleavage with the third HNH domain. DNA phosphorothioate in Rp configuration was specifically embedded in a hydrophobic cavity of ScoMcrA. This is the first structural report of the recognition of natural DNA phosphorothioate.