| As an important participant in the biological central dogma,helicase plays an important role in many fields in vivo,including DNA unwinding,gene repair and genic recombination so on.RecD is an important member of the SF1 B helicase family,and has been found widely in prokaryotic and eukaryotic organisms.It is divided into two classes.One is that require other subunits to assist in physiological activity,such as ReBCD.The other has been named RecD2 that is he Rec D-like proteins from organisms without Rec B and Rec C.A surprising exception is the RecD2 from Deinococcus radiodurans which is remark-ably resistant to ionizing radiation,ultraviolet light and other DNA-damaging agents.Its DNA can suffer many double-strand breaks.There has been much work on the physiological attributes,enzymes,and enzymatic pathways that contribute to its extraordinary ability to survive under extreme conditions.So far,many studies on enzyme kineticsin of RecD2 are only within DNA duplex.But other aspects have not been extensively explored.The interaction of RecD2 with specific DNA is not clear,especially G4 DNA that relates to ageing process,death and carcinogenesis.In this study,we got the RecD2 by prokaryotic expression and purification in terms of the correct pET22b-RecD2 plasmid from the cooperative laboratory.The binding and unwinding activity of RecD2 with specific DNA were analyzed by fluorescence anisotropy and stopped-flow fluorometric techniques,particularly on G4 DNA.It provides basis for the further research on the physiological function of RecD2,but also provided in-depth reference for the study on the similarities and differences among the helicase family.The main conclusions of this study are summarized as follows:1.In this study,we transformed the pET22b-RecD2 plasmid into Escherichia coli BL21.The cells were grown at 37?until OD600 0.6-0.8 then induced with 1mM IPTG for 3h.Purificatin was by 5mL HisTrap Ni and 5mL HisTrap Q with AKTA protein purification system.we got the 3mg of RecD2 helicase with purity greater than 95%.its molecular weight is about 76.38 kD.2.The first priority for kinetic characterization of RecD2 was determination of the binding affinity of RecD2 for substrate DNA.In the conditions of 20 mM Tris-HCl pH7.5,4m M MgCl2 and 1mM DTT,we found the binding affinity of RecD2 to ss16 nt DNA isinfluenced by different NaCl concentrations.50 mM is the best,and an average Km is 11.29 n M.3.Binding studies were performed by fluorescence anisotropy in order to characterize the affinity of RecD2 with single stranded DNA(ss16ntDNA),DNA duplex(ds 16 bp DNA)and G4 DNA.RecD2 can tightly bind to G4 DNA with 5'-single-stranded tail with Kd of13.96±0.034 nM.The binding affinity with single stranded DNA and G4 DNA is more than DNA duplex.4.when RecD2 bind to different G4 DNA,we found RecD2 express a preference for5'-single-stranded tail of G4 DNA in the conditions of 5mM Tris-HCl pH7.5,50 mM NaCl,2m M MgCl2 and 2 mM DTT.5.RecD2 can efficiently unwind 14 nt 5' overhang with 20 bp and 5'12nt fork with20 bp.An inverse association was observed between the unwinding activity and NaCl concentrations.The higher the concentration of salt,the weaker the activity of unwinding.6.We found RecD2 can unwind G4 DNA with rate of 5.268±0.38 s-1 in the appropriate conditions of 25 mM Tris-HCl pH7.5,50 mM NaCl,2mM MgCl2 and 2mM DTT with37?.Through single variable method,it was found that the effect of pH and temperature on unwinding activity is obvious.The rate of unwinding increases obviously with pH 7 and37?.7.Although the rate of RecD2 with 26 nt 5' G4 with 17 bp is higher than 26 nt overhang with 17 bp,the unwinding amplitude is much reduced.It indicates that G4 DNA may not be the natural substrates of RecD2,and the G4 structure has some block effect on the unwinding of RecD2 with DNA duplex.We purified RecD2 helicase and found it can bind and unwind G4 DNA.The study provides the basis for further research on the similarities and differences of the helicase family. |
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