Cleavage Of S-DAN By Phosphorothioation-Depedent Restriction Endonuclease SprMcrA And Structrual Basis For Its Sequence Specificity

DNA is more than just combinations of A,G,C,and T.Naturally occurring variations of the canonical nucleotides can be found in the DNA.A variety of chemical groups can be biologically appended to the nucleobase portion of a nucleotide.5-methylcytosine(^5mC)is the most in-depth studied,which is involved in DNA replication and gene regulation through the ⁵mm C recognition domain?SRA?,and is closely related to epigenetics.These DNA modifications are also referred to as epigenetic modifications.In addition,there is a unique type of DNA modification located on the DNA backbone and named phosphorothioation?PT?,in which the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur.In bacteria,epigenetic modifications have more specialized roles in DNA restriction and modification system?R-M system?.The R-M system can be divided into type I,II,III and IV according to the subunit composition,recognition sequence,cofactor requirement and cutting mode of the restriction enzyme and the modification enzyme.Type IV is also called as modification-dependent restriction system,of which the one can produce a clear and fixed cleavage band pattern in gel electrophoresis is now classified as a type IIM category.In the previous study of our lab,we identified a type IV endonuclease ScoMcrA from Streptomyces ceolicolor.ScoMcrA can recognize and cleave DNA with PT and ⁵mm C modification.The structure of ScoMcrA is resolved and reveals that it contains both a PT modification binding domain?SBD?and a ^5mC-binding domain?SRA-like?.ScoMcrA has the following characteristics:1)ScoMcrA only recognizes and cleaves the symmetric sequence(5?-G_PSGCC-3?),making it impossible to know the cutting direction and cutting frequency;2)ScoMcrA displays a low specificity of PT or Non-PT DNA?Star activity begins to appear when the reaction time exceeds 5 minutes?;3)The activity of ScoMcrA for cutting PT-DNA is poor.These characteristics make it difficult for us to study its enzymatic activity and cleavage mechanism in the future work.Therefore,we expect to find a simpler PT-dependent endonuclease with higher activity,different target sequence or low star activity to study its enzymatic activity and cleavage mechanism.With the amino acid sequence of SBD_Sco as query,more than 2761homologs containing SBD are identified and classified into three groups according to their molecular weight and domain arrangement:1)head-SBD-SRA-HNH;2)head-SRA-HNH;3)SRA-HNH.SprMcrA,a ScoMcrA homolog of much smaller size from Streptomyces pristinaespiralis,consists of 326 animo acids with an SBD-HNH didomain components.SprMcrA is a biologically active PT dependent endonuclease with a high restriction efficiency?>10³?to dnd cluster in vivo.Compared with ScoMcrA,the SBD domain of SprMcrA has a different binding activity in sequence specificity and affinity.1)SBD_Spr can bind to three natural PT-DNA core sequences?GGCC,GAAC and GATC?with fully-or hemi-PT modification,but can't bind other two?GTTC and CCA?;By contrast,SBD_Scoco only bind GGCC with PT modification.2)The binding affinity of SBD_Spr is higher than SBD_Sco to GpsGCC.Similar to SBD_Sco,SBD_Spr only recognized PT-DNA with R_P stereoisomer in the EMSA assay in vitro.Instead of a typical HNH motif in HNH homolos,SprMcrA has a rare HRH motif.Purified SprMcrA can cleaves PT-DNA when supplied with Mn²⁺or Co²⁺in vitro.The cleavage occurred mostly at N11/N12 or N13/N14 away from the G_PSA linkage on the 5?side and generated a double-strand breakage,but only about half of the G_PSAAC sites in the PT-pUC18 can be cleaved due to the sequence specificity of the C-terminal HNH motif?VBN?VB?.SprMcrA has a high specificity?16-fold in REase fidelity index?to distinguish PT and Non-PT DNA.The cutting direction and cutting sites of SprMcrA are fixed,so that SprMcrA can be classified to type IIM endonuclease.We also construct the mutation of SprMcrA_R284N284N through site-directed mutagenesis that makes the HRH motif to a conserved HNH motif.SprMcrA_R284N has an increasing cleavage activity about 30 folds to the wildtype and a different sequence specificity of the cleavage site.The mechanism of the changes in the cleavage activity of SprMcrA_R284N is discussed on the basis of protein modelling for the mutant and wildtype enzyme.In order to explain why SBD_Sco and SBD_Spr have a different sequence specificity for PT-DNA,the structure of SBD_Spr and PT-DNA complex was resolved in this study.The binding ability of SBD_Spr to PT-DNA is provided by three parts:a pocket that bind to the sulfur atom by hydrophobic van der Waals force;a‘base contact'loop inserted to the DNA major groove and interacted with DNA bases through hydrogen bonding force;two‘phosphate binding'loop interacted with DNA phosphates by electrostatic forces.Compared to the structure of SBD_Sco,we find that the SBD specificity to PT-DNA lies in the charge of‘phosphate binding'loop.We have demonstrated this hypothesis by site-directed mutagenesis of SBD_Sco.We constructed ScoMcrA_E156R/D157R,which gains the restriction ability against dnd cluster which modifies the G_PSAAC/G_PSTTC sequence through the restriction assay in vivo.In summary,we characterized the function and structure of the type IIM phosphorothioate-specific DNA restriction endonuclease SprMcrA.In this study,the sequce specificity and cleavage site of SprMcrA was identified,which deepened the understanding of PT-DNA dependent endonuclease family.We constructed a mutant with high cleavage activity,which paved the way for its future application in mapping PT-modification in bacterial genomes.The structure of SBD_Spr and PT-DNA complex was resolved,and the mechanism of SBD domain recognition specificity for PT-DNA sequence was clarified.In addition,sequence specificity of ScoMcrA was changed under the basis of structure,which provide a new idea for sequence specificity engineering of nucleic acid binding protein.