Characterization Of Sco5333,a Type Ⅳ Restriction Endonuclease From Streptomyces Coelicolor

Streptomycetes are gram-positive bacteria, which possess complex life cycle and capable of producing versatile secondary metabolites. Over two-thirds of naturally derived antibiotics in current use are produced by streptomycetes. A model strain, Streptomyces coelicolor was reported harboring at least four methyl-specific restriction endonucleases, making it hard for foreign methylated DNA’s entry, which pose obstacles for research on streptomycetes.We identified a type IV restriction endonuclease gene sco5333 which is located in the genomic island Gi-12 of S. coelicolor. The Sco5333 expression plasmid DNA cannot be introduced into dcm+ E. coli hosts. As well, the uptake efficiency of Dcm expression vector DNA into E. coli host harboring sco5333 gene decreased by over 90%.Sco5333 contains a N-terminal SRA domain(SET- and RING-associated domain) and a C-terminal HNH motif. The latter structure is embedded in a zinc finger motif composed of CX2CX36CX2 C.Site-directed mutagenesis analysis demonstrated that the in vivo restriction activity of Sco5333 requires cooperative action of both SRA and HNH structure. The in vitro binding activity is governed by SRA domain, while HNH motif is in charge of the in vitro cleavage.Sco5333 specifically binds to Dcm modified pUC18 plasmid DNA but not to non Dcm-modified DNA in vitro. Further investigation revealed that Sco5333 can bind to a 219 bp DNA modified by M.AluI, M.HhaI, M.HpaII, M.MspI, CpG, GpC and Dcm respectively. A systematic saturation mutagenesis was applied to the flanking bases of 5mC on a 55 bp DNA centered with a Dcm modification site, and used as the substrates for Sco5333 binding assay. Results showed that Sco5333 can bind 5mC in all DNA sequence contexts either on fully- or hemi-methylated DNA. Moreover, Sco5333 can bind to 5mC on single-strand DNA.Sco5333 shows weak cleavage activity on pUC18 plasmid DNA without methylation and sequence specificity.Zn2+ can significantly enhance the binding affinity of Sco5333 to 5mC DNA, but inhibit the cleavage activity of Sco5333 in presence of Mg2+ or Mn2+. However, Sco5333C252 D, in which the fourth cysteine of zinc finger is substituted by Asparate, showed DNA cleavage activity even in presence of Zn2+ and displayed higher activity than the wild type. Interestingly, activity of Sco5333C252 D in presence of Mg2+ or Mn2+ could also be repressed by Zn2+. We thereafter proposed a model in which how Zn2+ regulates DNA binding and cleavage activity of Sco5333 by competitively coordination to HNH ans zinc finger.We demonstrated that genomic island Gi-12 where sco5333 is preside is a mobile actinomycete integrative and conjugative element(AICE).In this thesis we demonstrated that Sco5333 is a type IV restriction endonuclease, and characterized its 5mC DNA binding activity and non-methyl and non-sequence specific cleavage activity. Due to the 100-discrimination of 5mC-binding from cytosine, and sequence flexibility, Sco5333 has potential to be developed into an instrumental enzyme for enrichment of 5mC DNA in epigenetic or carcinogenesis research.