厌氧棒菌Pif1解旋酶的表达纯化及解旋条件优化
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摘要
【目的】通过原核表达系统获得嗜热的厌氧棒菌(Anaerobaculum hydrogeniformans)DNA解旋酶Pif1(AnaPif1),研究其体外最佳解旋条件,为深入研究嗜热菌Pif1家族解旋酶工作机理提供参考。【方法】构建重组载体pET15b-sumo-AnaPif1,将重组载体转入表达菌株BL21(DE3)中诱导表达目的蛋白,经Ni-NTA柱和Heparin柱纯化最终获得全长AnaPif1蛋白;利用停流-荧光共振能量转移(FRET)技术,以pH值和Tris-HCl、NaCl、MgCl_2浓度及反应温度、能量供体为考察因素,以反应时间常数或速率常数为指标,摸索该蛋白在体外的最佳解旋条件、最佳能量供体以及底物的偏好性。【结果】成功构建了pET15b-sumo-AnaPif1重组表达载体,通过诱导表达,获得了纯度大于95%的全长AnaPif1蛋白,其大小为55ku,等电点为6.89。AnaPif1在体外的最佳解旋条件为:Tris-HCl(pH7.0)20mmol/L、NaCl 20mmol/L、MgCl_22mmol/L、反应温度37℃;其能够利用4种核苷三磷酸(ATP/GTP/CTP/UTP)作为能量供体,但对ATP有偏好性;该酶还能解旋多种底物(双链DNA、G4-DNA),且对底物选择无明显偏好性。【结论】获得了纯化的AnaPif1解旋酶,明晰了其最佳解旋条件、能量供体及底物的偏好性特征。
【Objective】The Pif1 helicase from Anaerobaculum hydrogeniformans(AnaPif1)was obtained by prokaryotic expression system,and the optimal conditions were explored for AnaPif1 to display unwinding activity in vitro.This research would be helpful for further study on the unwinding mechanism of Pif1 family helicases.【Method】The entire gene of AnaPif1 was synthesized and a recombinant plasmid pET15b-sumo-AnaPif1 was constructed.Then,it was transformed into Escherichia coli host strain BL21(DE3).The full-length AnaPif1 helicase was purified through Ni-NTA affinity chromatography followed by Heparin chromatography with fast protein liquid chromatography(FPLC)system.The stopped-flow and fluorescence resonance energy transfer(FRET)technique were used to investigate the effect of pH value and concentrations of Tris-HCl,NaCl,MgCl_2 as well as different temperature,energy donors and substrates on reaction activity of AnaPif1 in vitro.The time constant or rate constant were used as the determinant for optimal unwinding conditions for AnaPif1,selectivity of DNA substrates and energy donors.【Result】The recombinant plasmid pET15b-sumo-AnaPif1 was successfully constructed and the full-length AnaPif1heli-case with high purity(>95%)was obtained.The optimal unwinding conditions in vitro were Tris-HCl(pH 7.0)20mmol/L,NaCl 20mmol/L,MgCl_22mmol/L at 37 ℃.All nucleoside triphosphates could act as its energy donors,while ATP was preferred.AnaPif1 had no preference for DNA substrates such as G4-DNA and double-stranded DNA.【Conclusion】AnaPif1helicase was obtained,and the optimal unwinding conditions in vitro,energy donors and the preference of DNA substrates were determined.
【Objective】The Pif1 helicase from Anaerobaculum hydrogeniformans(AnaPif1)was obtained by prokaryotic expression system,and the optimal conditions were explored for AnaPif1 to display unwinding activity in vitro.This research would be helpful for further study on the unwinding mechanism of Pif1 family helicases.【Method】The entire gene of AnaPif1 was synthesized and a recombinant plasmid pET15b-sumo-AnaPif1 was constructed.Then,it was transformed into Escherichia coli host strain BL21(DE3).The full-length AnaPif1 helicase was purified through Ni-NTA affinity chromatography followed by Heparin chromatography with fast protein liquid chromatography(FPLC)system.The stopped-flow and fluorescence resonance energy transfer(FRET)technique were used to investigate the effect of pH value and concentrations of Tris-HCl,NaCl,MgCl_2 as well as different temperature,energy donors and substrates on reaction activity of AnaPif1 in vitro.The time constant or rate constant were used as the determinant for optimal unwinding conditions for AnaPif1,selectivity of DNA substrates and energy donors.【Result】The recombinant plasmid pET15b-sumo-AnaPif1 was successfully constructed and the full-length AnaPif1heli-case with high purity(>95%)was obtained.The optimal unwinding conditions in vitro were Tris-HCl(pH 7.0)20mmol/L,NaCl 20mmol/L,MgCl_22mmol/L at 37 ℃.All nucleoside triphosphates could act as its energy donors,while ATP was preferred.AnaPif1 had no preference for DNA substrates such as G4-DNA and double-stranded DNA.【Conclusion】AnaPif1helicase was obtained,and the optimal unwinding conditions in vitro,energy donors and the preference of DNA substrates were determined.
引文
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[17]Zhang D H,Zhou B,Huang Y,et al.The human Pif1helicase,apotential Escherichia coli RecD homologue,inhibits telomerase activity[J].Nucleic Acids Research,2006,34(5):1393-1404.
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[19]Bailey S,Eliason W K,Steitz T A.The crystal structure of the Thermus aquaticus DnaB helicase monomer[J].Nucleic Acids Research,2007,35(14):4728-4736.
[20]Fan L,Fuss J O,Cheng Q J,et al.XPD helicase structures and activities:insights into the cancer and aging phenotypes from XPD mutations[J].Cell,2008,133(5):789-800.
[21]Zhang X D,Dou S X,Xie P,et al.Escherichia coli RecQ is a rapid,efficient,and monomeric helicase[J].The Journal of Biological Chemistry,2006,281(18):12655-12663.
[22]Chen W F,Dai Y X,Duan X L,et al.Crystal structures of the BsPif1helicase reveal that a major movement of the 2BSH3domain is required for DNA unwinding[J].Nucleic Acids Research,2016,44(6):2949-2961.
[23]Zhou X,Ren W.Structural and functional insights into the unwinding mechanism of Bacteroides sp Pif1[J].Cell Reports,2016,14(8):2030-2039.
[24]Liu N N,Duan X L,Ai X,et al.The Bacteroides sp.3_1_23Pif1protein is a multifunctional helicase[J].Nucleic Acids Research,2015,43(18):8942-8954.
[25]Duan X L,Liu N N,Yang Y T,et al.G-quadruplexes significantly stimulate Pif1helicase-catalyzed duplex DNA unwinding[J].Journal of Biological Chemistry,2015,290(12):7722-7735.
[2]Abdel-Monem M,Hoffmann-Berling H.Enzymic unwinding of DNA:purification and characterization of a DNA-dependent ATPase from Escherichia coli[J].European Journal of Biochemistry,1976,65(2):431-440.
[3]Jankowsky E.RNA helicases at work:binding and rearranging[J].Trends in Biochemical Sciences,2011,36(1):19-29.
[4]Gorbalenya A E,Koonin E V.Helicases amino acid sequence comparisons and structure function relationships[J].Current Opinion in Structural Biology,1993,3(3):419-429.
[5]Lohman T M,Ferrari M E.Escherichia coli single-stranded DNA binding protein:multiple DNA binding modes and cooperativities[J].Annual Review of Biochemistry,1994,63:527-570.
[6]Deng Z,Lehmann K C,Li X,et al.Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase[J].Nucleic Acids Research,2014,42(5):3464-3477.
[7]Foury F,Kolodynski J.Pif mutation blocks recombination between mitochondrial rho+and rho-genomes having tandemly arrayed repeat units in Saccharomyces cerevisiae[J].Proc Natl Acad Sci USA,1983,80(17):5345-5349.
[8]Lahaye A,Stahl H,Thines-sempoux D,et al.PIF1:a DNA helicase in yeast mitochondria[J].Embo J,1991,10(4):997-1007.
[9]Boule J B,Vega L R,Zakian V A.The yeast Pif1p helicase removes telomerase from telomeric DNA[J].Nature,2005,438(7064):57-61.
[10]Budd M E,Reis C C,Smith S,et al.Evidence suggesting that Pif1helicase functions in DNA replication with the Dna2helicase/nuclease and DNA polymerase delta[J].Molecular and Cellular Biology,2006,26(7):2490-2500.
[11]Boule J B,Zakian V A.The yeast Pif1p DNA helicase preferentially unwinds RNA-DNA substrates[J].Nucleic Acids Research,2007,35(17):5809-5818.
[12]Azvolinsky A,Dunaway S,Torres J Z,et al.The S.cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes[J].Genes&Development,2006,20(22):3104-3116.
[13]Bessler J B,Zakian V A.The amino terminus of the Saccharomyces cerevisiae DNA helicase Rrm3p modulates protein function altering replication and checkpoint activity[J].Genetics,2004,168(3):1205-1218.
[14]Schmidt K H,Derry K L,Kolodner R D.Saccharomyces cerevisiae RRM3,a 5′to 3′DNA helicase,physically interacts with proliferating cell nuclear antigen[J].The Journal of Biological Chemistry,2002,277(47):45331-45337.
[15]Wang Q,Liu J Q,Chen Z,et al.G-quadruplex formation at the3′end of telomere DNA inhibits its extension by telomerase,polymerase and unwinding by helicase[J].Nucleic Acids Research,2011,39(14):6229-6237.
[16]Futami K,Shimamoto A,Furuichi Y.Mitochondrial and nuclear localization of human Pif1helicase[J].Biological&Pharmaceutical Bulletin,2007,30(9):1685-1692.
[17]Zhang D H,Zhou B,Huang Y,et al.The human Pif1helicase,apotential Escherichia coli RecD homologue,inhibits telomerase activity[J].Nucleic Acids Research,2006,34(5):1393-1404.
[18]Singleton M R,Scaife S,Raven N D,et al.Crystallization and preliminary X-ray analysis of RecG,a replication-fork reversal helicase from Thermotoga maritima complexed with a threeway DNA junction[J].Acta crystallographica Section D Biological Crystallography,2001,57(Pt 11):1695-1696.
[19]Bailey S,Eliason W K,Steitz T A.The crystal structure of the Thermus aquaticus DnaB helicase monomer[J].Nucleic Acids Research,2007,35(14):4728-4736.
[20]Fan L,Fuss J O,Cheng Q J,et al.XPD helicase structures and activities:insights into the cancer and aging phenotypes from XPD mutations[J].Cell,2008,133(5):789-800.
[21]Zhang X D,Dou S X,Xie P,et al.Escherichia coli RecQ is a rapid,efficient,and monomeric helicase[J].The Journal of Biological Chemistry,2006,281(18):12655-12663.
[22]Chen W F,Dai Y X,Duan X L,et al.Crystal structures of the BsPif1helicase reveal that a major movement of the 2BSH3domain is required for DNA unwinding[J].Nucleic Acids Research,2016,44(6):2949-2961.
[23]Zhou X,Ren W.Structural and functional insights into the unwinding mechanism of Bacteroides sp Pif1[J].Cell Reports,2016,14(8):2030-2039.
[24]Liu N N,Duan X L,Ai X,et al.The Bacteroides sp.3_1_23Pif1protein is a multifunctional helicase[J].Nucleic Acids Research,2015,43(18):8942-8954.
[25]Duan X L,Liu N N,Yang Y T,et al.G-quadruplexes significantly stimulate Pif1helicase-catalyzed duplex DNA unwinding[J].Journal of Biological Chemistry,2015,290(12):7722-7735.