Expression,Purification And Enzyme Activity Analysis Of OrcDHX36 Helicase

The special physiological activity of helicases plays a significant role in the development of living organism.Helicases are widely involved in the replication,transcription,translation and repair of genetic information,RNA splicing and telomere elongation.DHX36 helicase(also known as RHAU,G4R1),is one of the DEAH-box subfamily in the SF2 superfamily.Previous studies have shown that DHX36 are important in different biological processes,such as hematopoiesis,cardiac development,spermatogonial differentiation and Multiple immune responses.Currently,the biological functions of the C-terminal domain are poorly understood,but a large number of studies have shown that the N-terminal domain plays a crucial role in the binding of DHX36 to the G4 structure.However,the molecular mechanism of DHX36 is still unknown due to the lack of structural information of the N-terminal domain.In this paper,Orcinus orca DHX36 is choosen as the research object.Based on the principles of fluorescence anisotropy and fluorescence resonance energy transfer,gel electrophoresis,fluorescence anisotropy and rapid stop-current technology are used.The substrate binding activity,unwinding activity in vitro and difference enzymatic activity between homologous proteins were measured and compared systematically.The main experimental results are as follows:(1)The recombinant plasmid pET-21a-HMT-OrcDHX36 was constructed with gene of the DHX36 protein,and induced by expression of Ecoli.C2566H.The target protein with good solubility was obtained.OrcDHX36 with high purity and activity was obtained by using maltose affinity medium and cation exchange column.(2)OrcDHX36 has obvious substrate preference and can specifically recognize and bind to the structure of G4 DNA and G4 RNA.However,unlike the reported homologous proteins,OrcDHX36 cannot bind to ss DNA and ds DNA,and has no sequence binding specificity.(3)OrcDHX36 has a 3'-5'unwinding direction,and has a specific unwinding activity for G4 DNA,but not for ds DNA.The unwinding ability of OrcDHX36 on G4 DNA is affected by the 3'over hang.(4)The optimal unwinding conditions of OrcDHX36 in vitro were ascertained.Buffer pair:Tris-HCl,p H 7.5,30 m M Na Cl,1 m M Mg Cl₂,1 m M DTT,protein concentration 300n M,reaction temperature 25°C.(5)The existence of a large number of DHX36 without RSM,including OrcDHX36protein,was identified clearly.The phylogenetic tree of DHX36 homologous protein was constructed by the Neighbor-joining method to clarify the sequence characteristic relationship of DHX36.(6)The amino acid sequence and helicase characteristic activity of DHX36 without RSM region including OrcDHX36,Mel DHX36,Cl DHX36 and Cl DHX36^91-968 were analyzed and compared.The results showed that both OrcDHX36 and Mel DHX36 exhibited high binding and unwinding activity against G4 structure.The activity does not depend on the dozens of acid sequence at the N terminal.However,the unwinding activity of Cl DHX36 and its enzyme-digested product,Cl DHX36^91-968,indicated that Cl DHX36 depended on an amino acid sequence similar to the RSM domain.To sum up,this study reported the construction,expression and purification process of ORCDHX36 for the first time,measured and analyzed the characteristic activity of the protein helicase,and obtained the optimal conditions of in vitro helicase.Differ from previous reports on DHX36 with complete RSM region,we identified that DHX36 helicases have three kinds of characteristic N-terminal amino acid sequences in this study.Although the sequences are different,all of them have specific binding and unwinding activities on the G4 structure.