Expression,purification And Enzyme Activity Analysis Of ItWRN Helicase

Helicases are a type of molecular motor proteins that use energy derived from ATP hydrolysis to separate complementary strands of nucleic acid duplexes.The helicase Rec Q is highly conserved in both prokaryotes to eukaryotes and plays a key role in maintaining the stability of the body and protecting the genome from deleterious effects.WRN belongs to a class of DNA helicases in the Rec Q subfamily of the helicase superfamily II.Lack of WRN(Werner syndrome protein),BLM(Bloom syndrome protein),and RECQ4,among the five types of Rec Q helicases found in human cells,lead to rare recessive genetic disorders,Werner,Bloom,and Rothmund-Thomson syndrome,respectively.These diseases are often associated with cancer susceptibility and accelerated aging,and patients exhibit high levels of genomic instability such as sister chromatid exchange and abnormal telomere shortening.Previous studies have found that WRN is involved in the unfolding process of the G4 structure formed in DNA replication leading strand.Recent studies have demonstrated that cells lacking WRN cannot promote repair of non-homologous terminal junction-mediated double-strand breaks.Only by restoring the activities of WRN lysozyme and nuclease can this be remedied.WRN also interacts with other DNA repair proteins to participate in base excision repair and promote replication of replication forks at sites of DNA damage or after replication fork arrest.Its multiple interactions with key proteins in DNA replication,repair,recombination and telomere maintenance suggest that WRN has a coordinating function with these processes in human cells.In this study,Ictidomys tridecemlineatus WRN helicase(ItWRN^501-1280)was selected as the research object by bioinformatics analysis and sequence alignment.The sequence was listed as the core domain fragment and the sequence was stable,so it could be used as the purified protein.The state of protein aggregation was determined by gel filtration chromatography and dynamic light scattering experiment,and the enzymatic characteristics were studied by fluorescence anisotropy and stopped-flow fluorescence energy transfer technique.Finally,the complex crystals of ItWRN and G4 DNA were tested and screened with the activity experiment.The main experimental results are as follows:(1)The recombinant plasmid p ET-15b-sumo-ItWRN was obtained by in vitro recombination and expressed in E.coli by low temperature induction.The soluble target protein was obtained by purification methods such as affinity chromatography,gel filtration chromatography and ultrafiltration,and the purity was more than 90%.The gel filtration chromatography and dynamic light scattering measurements show that the aggregation state of the gel in the solution is monomer,with good homogeneity.(2)The determination of the binding activity of ItWRN to single and double-stranded substrates shows that the enzyme is dependent on the length of the tail chain.The longer the ss DNA chain,the stronger the binding activity;and the binding activity to 3'overhang ds DNA is stronger than the blunt end ds DNA.The longer the blunt-ended ds DNA chain,the weaker its binding activity.Binding experiments on sequence-specific substrates showed that ItWRN tended to bind to guanine and cytosine.In addition,ItWRN showed high binding activity to G4 DNA and Fork DNA with different substrate structure specificities.The determination of the substrate unwinding activity of ItWRN shows that its unwinding polarity was 3'-5'.Moreover,the enzyme had some certain unwinding ability to Fork DNA,ds DNA and G4 DNA.(3)The gel filtration chromatography was used to study the verification experiment of the complex of ItWRN and G4 DNA.The enzyme is complexed with G4 with tail lengths of7T/8T/9T,and the minimum tail length required for binding to the substrate was 8T,the optimal tail chain length to form a complex was 9T.In addition,the verification experiment of the complex of ItWRN and bipolar G4 DNA showed that there are two states of monomer complex and dimer complex,and mainly exist in the form of dimer complex.In conclusion,we have successfully constructed and purified the highly purified ItWRN helicase,clarified its aggregation state and homogeneity in solution,and analyzed its binding activity and unwinding preference to different nucleic acid substrates.Finally,the complex state of the enzyme after compounding with G4 DNA was confirmed by complex verification experiments,which laid a solid foundation for the study of three-dimensional structure of the Rec Q family helicase.