| Helicases are a class of ubiquitous molecular motor proteins found in all organisms thatparticipate in almost all aspects of the DNA metabolism. Generally， helicases manifest anATP binding and hydrolysis activity which is depended on the presence of DNA. Bycoupling the energy of ATP hydrolysis to the breakage of hydrogen bonds between duplexoligonucleotides, helicases provide the single strand DNA as templates or reactionintermediates which are required for DNA replication, transcription, repair and recombination.Different helicases share the conserved amino acid motifs. According to the diversity of thenumber and sequence of the motifs, helicase were classified into five super family. Based onthe directivity of unwinding reaction, helicases can also be divided into two types:5’－3’ or3’－5’ direction. It has been confirmed that the mutations of helicase genes always lead toserious genetic disease, such as Werner, Bloom and Fanconi syndrome, these findings makehelicase attracting an increasing attention.Pif1helicase family is a class of ATP-dependent,5’-3’ directed helicases which belongto helicase Super FamilyⅠ, and widely exist in prokaryotic, eukaryotic and viruses. In vivo,Pif1helicases affect the replication of telomere, rDNA and mitochondrial DNA, involves inthe processing of Okazaki fragment, play an important role in maintaining the stability ofthe chromosome and mitochondrial DNA. However, the structure and mechanism of Pif1helicase family remains unclear. In this study, we constructed the expression vector of DePif1,a Pif1helicase from thermophilic Deferribacter desulfuricans, based on expression vectorpET15b. Then, we explored the high-efficiency scheme for the expression and purification ofDePif1in E. coli expression system. Finally, the DNA binding and unwinding activities ofDePif1were analyzed. The main conclusions of this study are summarized as follows:1. Based on expression vector pET15b, we obtained the recombinant plasmidpET15b-SUMO-DePif1by introducing SUMO tag to the downstream of its His-tag. So,the recombinant DePif1fused His-tag and SUMO-tag orderly at its N-terminus. Thisplasmid was introduced into E. coli BL21(DE3) strain, induced and cultured at28℃.At this condition, recombinant DePif1has an high-efficiency expression and mostly exist in soluble form.2. DePif1with His6-SUMO tag was capture by affinity chromatography with a Ni-NTAcolumn firstly. Then, the fusion tag was cleaved by SUMO protease and tag-free DePif1helicase was obtained by further purification with Heparin Sepharose Fast Flow andNi-NTA chromatography. According to this protocol,9mg DePif1with>95%puritycould be obtained per liter culture.3. The DNA binding properties of DePif1was analyzed by Fluorescence anisotropy. Wefound, pH6.0,50mM NaCl, is the appropriate condition for the binding of DePif1withDNA substrates. At this condition, DePif1can well bind to ss-DNA, ds-DNA, G4-DNA,and the Kmvalue of these three substrates is3.02±0.65、18.82±1.70、2.71±0.57,respectively. This reveals that the binding strength of DePif1with different substrates isG4-DNA> single-stranded DNA> double-stranded DNA.4. The DNA unwinding activity of DePif1was studied by stopped-flow based on FRET.The results show that at40℃, in the presence of ATP and Mg2+conditions, DePif1can unwind double-stranded DNA and G4-DNA substrates effectively. However, theunwinding activity of DePif1to G4-DNA substrate is much higher than thedouble-stranded DNA substrate.In this study, We expressed and obtained purified DePif1helicase successfully. Activityanalysis of DePif1indicates that it can bind and unwind G4-DNA specially. These will laythe foundation for the elucidating of the structure and function of Pif1family helicase... |
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