SET and RING-associated domain-mediated dimerization in type IV restriction enzyme ScoMcrA promotes the affinity of sulfur binding domain to sulfur modified DNA

Abstract:

[Background] Some bacterial DNA skeleton undergo phosphorothioate modification, sulfur binding domain (SBD) can specifically recognize this physiological modification. Unlike most SBD-HNH di-domain nucleases, SET and RING-associated (SRA) domain, specifically recognize DNA 5-methylcytosine (5mC), is inserted between SBD and HNH domains of ScoMcrA. The crystal structures show that single SBD is a monomer and SBD-SRA is a dimer.[Objective] The effects of the presence of SRA domain on the binding of sulfur modified DNA by SBD, and the way SRA domain affect the phosphorothioated DNA recognition. [Methods] Electrophoresis mobility shift assay (EMSA) was applied to compare binding affinity of SBD and SBD-SRA to sulfur modified DNA respectively. The key amino acid residues involved in dimerization of SBD-SRA were mutated to examine the binding affinity of mutant proteins to phosphorothioated DNA. [Results] Compared with SBD domain alone, the di-domain protein SBD-SRA showed enhanced affinity to sulfur modified DNA. The single point mutation of ten amino acid residues at the dimer-forming interface of SBD and SRA domain seldom affect its binding affinity to sulfur modified DNA. By comparison, L₂₆₁LGET₂₆₅ are simultaneously mutated to A₂₆₁AAAA₂₆₅ on SBD-SRA, the binding affinity of the mutant to sulfur modified DNA decreased to a level similar to that of SBD. [Conclusion] According to EMSA results, we primarily came to the conclusion that SRA domain can improve the binding ability of SBD to sulfur modified DNA in SBD-SRA di-domain protein; L₂₆₁LGET₂₆₅ is the key amino acid sites in the SRA domain that affects the binding ability of SBD to sulfur modified DNA.