Expression, Purification Andphysicochemical Properties Analysisof Bos Taurus RECQL Helicase

Helicases are ubiquitous molecular motors that unwind double or multi-stranded DNA or RNA structures in a 3’â†'5’ or 5’â†'3’ directionality by using ATP as an energy source. They play critical roles in chromosomal and plasmid replication, transcription, translation, DNA recombination and repair. RECQ DNA helicases are SFII enzymes conserved from prokaryotes to mammals that play a key role in the maintenance of genome stability. RECQL is the first RECQ helicase that was discovered, owing to its strong ATPase activity and its high abundance in cells. It plays an essential role in maintaining the stability of genome stability, and participates in chromosome replication, DNA repair and telomere maintenance.Although have achieved some achievements on RECQL helicase, characterized in terms of enzymatic activity and function is not yet clearly, kinetics of enzyme catalyzed reaction,structure and mechanism of RECQL helicase remains unclear.In this study, the full-length, truncated and mutational RECQL helicase of Bos taurus were expressed and purified in Escherichia coli, analyzed the state and DNA binding activity of BtRECQLTM, optimized unwinding conditions of BtRECQL, compared the unwinding activity of BtRECQL, BtRECQLT, BtRECQLTM on different substrates under the optimum unwinding conditions. The concrete results of this study as followes:1. We synthesised the full-length coding sequence of BtRECQL that has been optimized,and constructed the recombinant plasmid p ET21a-Bt Recql successfully. Then, the recombinant plasmid was transformed into E. coli host strain BL21(DE3) and induced to expression. BtRECQL proteins were purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography(Superdex 200) on FPLC system. Finally, we obtained about0.29 mg full-length BtRECQL with purity above 95% from 1L culture medium.2. We constructed the truncated recombinant plasmid p ET15b-Bt Recql T on the basis of full-length coding sequence. Then, the truncated protein BtRECQLT was induced to expression in E. coli host strain BL21(DE3). BtRECQLT proteins were purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography(Superdex 200) on FPLC system. Through the above steps, 3.1 mg truncated protein BtRECQLT could be obtained with purity above 95% from 1L culture medium.3. Based on the sequence of BtRECQLT, we constructed the mutant recombinant plasmid p ET15b-Bt Recql TM. BtRECQLTM was induced to expression in E. coli host strain BL21(DE3),then, using the purification methods as BtRECQLT, 0.84 mg mutant proteins with purity above 90% could be obtained from 1L culture medium.4. Regardless of whether the presence of ATP or DNA, We found BtRECQLTM existed as monomer formation by dynamic light scattering experiment. Analyzed the binding activity of BtRECQLTM with different substrates by Fluorescence anisotropy, we found the binding ability of BtRECQLTM with ss DNA was higher than ds DNA. Moreover, we also found that the binding ability of BtRECQLTM gradually increased with the growth of chain for the same type substrate. For G4 DNA, with tail substrates are more likely bound with BtRECQLTM than without tail.5. Through analyzing unwinding process of BtRECQL using fluorescence anisotropy and various stopped-flow assays, we found under the conditions that Bt Recql was 60 nmol/L, ATP concentration was 1 mmol/L, reaction temperature was 37℃ and the reaction buffer was 30mmol/L Tris-HCl p H7.5, 60 mmol/L Na Cl, 1 mmol/L Mg Cl2, 2 mmol/L DTT, BtRECQL displayed the strongest unwinding activity.6. Under the optimum conditions, we analyzed the unwinding activity of BtRECQL,BtRECQLT, BtRECQLTM to ds DNA and G4 DNA, the results showed that the unwinding activity of BtRECQLT was strongest no matter the substrate was ds DNA or G4 DNA, and BtRECQL was weakest. The results indicated that the dimer state of RECQL helicase contribute to the unwinding activity, and the mixture of different oligomeric forms against the unwinding activity.In this study, BtRECQL, BtRECQLT, BtRECQLTM were expressed and purified successfully, and analyzed the physicochemical properties of BtRECQLTM. Optimized the unwinding conditions of BtRECQL, and under the optimum conditions, we analyzed the unwinding activity of BtRECQL, BtRECQLT, BtRECQLTM to ds DNA and G4 DNA. Our research can provide a reference for the further study of structure and function of RECQL helicase...