嗜热厌氧杆菌Ana.Pif1解旋酶核心结构域蛋白与DNA底物结合的反应特性
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摘要
目的:探究嗜热厌氧杆菌的Ana. Pif1解旋酶核心结构域蛋白(Ana. Pif1-HD)与DNA底物结合的反应特性。方法:利用DNAman、Cluxtal X等软件,对Ana. Pif1的核心结构域蛋白进行系统进化树分析与同源序列比对; Ana. Pif1-HD表达载体构建后转入大肠杆菌进行原核表达,并通过Ni-NTA、SUMO酶切、S200分子筛等层析柱纯化获得Ana. Pif1-HD蛋白;利用Stopped-flow FRET技术监测Ana. Pif1-HD蛋白的解旋活性,利用荧光偏振检测技术分析Ana. Pif1-HD与不同长度、不同结构DNA底物的结合反应各向异性值,并进行拟合分析与统计。结果:Ana. Pif1的核心结构域蛋白为靠近N-端的448氨基酸,通过原核表达与纯化获得纯度为97%、浓度为5 g/L的Ana. Pif1-HD蛋白,并利用Stopped-flow FRET与荧光偏振等技术验证其具有Ana. Pif1全长蛋白90%的解旋活性与95%的结合活性; Ana. Pif1-HD的最佳结合反应条件是42℃在pH 6. 0含20 mmol/L Na Cl及3 mmol/L MgCl2的溶液中进行; Ana. Pif1-HD与不同长度ss DNA底物结合时的解离速率常数递增,并明确其ssDNA的binding-size为10. 34 nt;研究首次揭示Ana. Pif1-HD与不同复制中间体DNA结合反应的底物特异性ssG4 DNA> G4 DNA> 3'-ssDNA-dsDNA≈Y-型>其它底物。结论:成功表达纯化具有全长活性的Ana. Pif1解旋酶核心结构域蛋白,首次揭示该解旋酶核心蛋白与各类不同DNA底物的结合反应特性。
Objective: To explore the characteristics of binding reactions of Anaerobaculum hydrogeniforman Pif1 helicase's core domain(Ana. Pif1-HD). Methods: The conserved Ana. Pif1-HD was analyzed through homology sequence alignment and phylogenetic tree by bioinformatics software DNAman and CluxtalX. The constructed Ana. Pif1-HD was inducible expressed in E. coli,and purified through Ni-NTA,SUMO protease cleaved,Superdex 200 gel filtration chromatography. Stopped-flow FRET was used to detect the unwinding activity of Ana. Pif1-HD,and the characteristics of such helicase binding reactions were mainly measured by fluorescence polarization technique. Result: The Ana. Pif1-HD protein was confirmed as 448 aa in the N-terminal region. By inducible expression and purification,Ana. Pif1-HD protein with 97% purity and 5 mg/m L concentration was obtained,and this purified product was confirmed with above 90% unwinding activity and 95% binding activity. The optimum binding conditions of Ana. Pif1-HD were pH = 6. 0 PBS buffer contains 20 mmol/L NaCl and3 mmol/L MgCl2 under 42 ℃. Our research showed the dissociation rate constant(Kd) gradually increased with the increase in length of ss DNA,and the binding-size of Ana. Pif1-HD with ss DNA was10. 34 nt. Furthermore,the DNA-binding substrates specificity of Ana. Pif1-HD was revealed that ssG4 DNA > G4 DNA > 3'-ssDNA-dsDNA≈Y-structure > Other substrates. Conclusions: The Ana. Pif1-HD with Ana. Pif1-full protein's activity is successfully purified,and this research systematically analyzes the binding characteristics of the Ana. Pif1 helicase for the frist time. It is helpful in providing good experimental base for the further elucidation of the molecular mechanism of thermophiles' helicases.
Objective: To explore the characteristics of binding reactions of Anaerobaculum hydrogeniforman Pif1 helicase's core domain(Ana. Pif1-HD). Methods: The conserved Ana. Pif1-HD was analyzed through homology sequence alignment and phylogenetic tree by bioinformatics software DNAman and CluxtalX. The constructed Ana. Pif1-HD was inducible expressed in E. coli,and purified through Ni-NTA,SUMO protease cleaved,Superdex 200 gel filtration chromatography. Stopped-flow FRET was used to detect the unwinding activity of Ana. Pif1-HD,and the characteristics of such helicase binding reactions were mainly measured by fluorescence polarization technique. Result: The Ana. Pif1-HD protein was confirmed as 448 aa in the N-terminal region. By inducible expression and purification,Ana. Pif1-HD protein with 97% purity and 5 mg/m L concentration was obtained,and this purified product was confirmed with above 90% unwinding activity and 95% binding activity. The optimum binding conditions of Ana. Pif1-HD were pH = 6. 0 PBS buffer contains 20 mmol/L NaCl and3 mmol/L MgCl2 under 42 ℃. Our research showed the dissociation rate constant(Kd) gradually increased with the increase in length of ss DNA,and the binding-size of Ana. Pif1-HD with ss DNA was10. 34 nt. Furthermore,the DNA-binding substrates specificity of Ana. Pif1-HD was revealed that ssG4 DNA > G4 DNA > 3'-ssDNA-dsDNA≈Y-structure > Other substrates. Conclusions: The Ana. Pif1-HD with Ana. Pif1-full protein's activity is successfully purified,and this research systematically analyzes the binding characteristics of the Ana. Pif1 helicase for the frist time. It is helpful in providing good experimental base for the further elucidation of the molecular mechanism of thermophiles' helicases.
引文
[1]WONG I,LOHMAN T M. Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding[J]. Science,1992,256(5055):350-355.
[2]BIANCO P R,KOWALCZYKOWSKI S C. Translocation step size and mechanism of the Rec BC DNA helicase[J].Nature,2000,405(6784):368-372.
[3]SCHLIERF M,HA T. A helicase with an extra spring in its step[J]. Cell,2012,151(2):244-246.
[4]BOCHMAN M L,JUDGE C P,ZAKIAN V A. The Pif1family in prokaryotes:what are our helicases doing in your bacteria?[J]. Mol Biol Cell,2011,22(12):1955-1959.
[5]BYRD A K,RANEY K D. Structure and function of Pif1helicase[J]. Biochem Soc Trans,2017,45(5):1159-1171.
[6]CHUNG W H. To peep into Pif1 helicase:multifaceted all the way from genome stability to repair-associated DNA synthesis[J]. J Microbiol,2014,52(2):89-98.
[7]WILSON M A,KWON Y,XU Y,et al. Pif1 helicase and Polδpromote recombination-coupled DNA synthesis via bubble migration[J]. Nature,2013,502(7471):393-396.
[8]DUAN X L,LIU N N,YANG Y T,et al. G-quadruplexes significantly stimulate Pif1 helicase-catalyzed duplex DNA unwinding[J]. J Biol Chem,2015,290(12):7722
[9]PAESCHKE K,BOCHMAN M L,GARCIA P D,et al.Pif1 family helicases suppress genome instability at Gquadruplex motifs[J]. Nature,2013,497(7450):458-462.
[10]HOU X M,WU W Q,DUAN X L,et al. Molecular mechanism of G-quadruplex unwinding helicase:sequential and repetitive unfolding of G-quadruplex by Pif1 helicase[J]. Biochem J,2015,466(1):189-199.
[11]STINUS S,PAESCHKE K,CHANG M. Telomerase regulation by the Pif1 helicase:a length-dependent effect[J]. Current Genetics,2018,64(2):509-513.
[12]LU KY,CHEN WF,RETY S,et al. Insights into the structural and mechanistic basis of multifunctional S.cerevisiae Pif1p helicase[J]. Nucleic Acids Res,2018,46(3):1486-1500.
[13]LIU N N,DUAN X L,AI X,et al. The Bacteroides sp.3_1_23 Pif1 protein is a multifunctional helicase[J].Nucleic Acids Res,2015,43(18):8942-8954.
[14]WEI X B,ZHANG B,BAZEILLE N,et al. A 3'-5'exonuclease activity embedded in the helicase core domain of Candida albicans Pif1helicase[J]. Sci Rep,2017,7:42865.
[15]SABOURI N. The functions of the multi-tasking Pfh1Pif1helicase[J]. Curr Genet,2017,63(4):621-626.
[16]郭海磊,刘娜女,段晓雷,等.厌氧棒菌Pif1解旋酶的表达纯化及解旋条件优化[J].西北农林科技大学学报(自然科学版),2017,45(6):206-212.
[17]段晓雷,刘娜女,付怡欣,等.嗜热脱铁去硫弧菌Pif1解旋酶生物学活性的分子特征分析[J].中国生物化学与分子生物学报,2018,34(1):77-88.
[18]MAUNE M W,TANNER R S. Description of Anaerobaculum hydrogeniformans sp. nov.,an anaerobe that produces hydrogen from glucose,and emended description of the genus Anaerobaculum[J]. Int J Syst Evol Microbiol,2012,62(Pt 4):832-838.
[19]SANDERS C M. Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity[J]. Biochem J,010,430(1):119-128.
[20]DONATI E R,CASTRO C,URBIETA M S. Thermophilic microorganisms in biomining[J]. World J Microbiol Biotechnol,2016,32(11):179.
[21]王帅锋,刘娜女,段晓雷,等.热脱硫弧菌解旋酶基因Ty Pif1的原核表达、纯化及功能分析[J].西北农林科技大学学报(自然科学版),2016,44(9):207-212.
[22]GEORGE T,WEN Q,GRIFFITHS R,et al. Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks[J]. Nucleic Acids Res,2009,37(19):6491-6502.
[23]OSMUNDSON J S,KUMAR J,YEUNG R,et al. Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes[J]. Nat Struct Mol Biol,2017,24(2):162-170.
[24]SINGH S P,KOC K N,STODOLA J L,et al. A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End[J]. J Mol Biol,2016,428(6):1053-1067.
[25]骆衡,许厚强,陈祥,等.荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作[J].生物化学与生物物理进展,2013,40(3):255-265.
[26]PATEL D S,MISENKO S M,HER J,et al. BLM helicase regulates DNA repair by counteracting RAD51 loading at DNA double-strand break sites[J]. J Cell Biol,2017,216(11):3521-3534.
[2]BIANCO P R,KOWALCZYKOWSKI S C. Translocation step size and mechanism of the Rec BC DNA helicase[J].Nature,2000,405(6784):368-372.
[3]SCHLIERF M,HA T. A helicase with an extra spring in its step[J]. Cell,2012,151(2):244-246.
[4]BOCHMAN M L,JUDGE C P,ZAKIAN V A. The Pif1family in prokaryotes:what are our helicases doing in your bacteria?[J]. Mol Biol Cell,2011,22(12):1955-1959.
[5]BYRD A K,RANEY K D. Structure and function of Pif1helicase[J]. Biochem Soc Trans,2017,45(5):1159-1171.
[6]CHUNG W H. To peep into Pif1 helicase:multifaceted all the way from genome stability to repair-associated DNA synthesis[J]. J Microbiol,2014,52(2):89-98.
[7]WILSON M A,KWON Y,XU Y,et al. Pif1 helicase and Polδpromote recombination-coupled DNA synthesis via bubble migration[J]. Nature,2013,502(7471):393-396.
[8]DUAN X L,LIU N N,YANG Y T,et al. G-quadruplexes significantly stimulate Pif1 helicase-catalyzed duplex DNA unwinding[J]. J Biol Chem,2015,290(12):7722
[9]PAESCHKE K,BOCHMAN M L,GARCIA P D,et al.Pif1 family helicases suppress genome instability at Gquadruplex motifs[J]. Nature,2013,497(7450):458-462.
[10]HOU X M,WU W Q,DUAN X L,et al. Molecular mechanism of G-quadruplex unwinding helicase:sequential and repetitive unfolding of G-quadruplex by Pif1 helicase[J]. Biochem J,2015,466(1):189-199.
[11]STINUS S,PAESCHKE K,CHANG M. Telomerase regulation by the Pif1 helicase:a length-dependent effect[J]. Current Genetics,2018,64(2):509-513.
[12]LU KY,CHEN WF,RETY S,et al. Insights into the structural and mechanistic basis of multifunctional S.cerevisiae Pif1p helicase[J]. Nucleic Acids Res,2018,46(3):1486-1500.
[13]LIU N N,DUAN X L,AI X,et al. The Bacteroides sp.3_1_23 Pif1 protein is a multifunctional helicase[J].Nucleic Acids Res,2015,43(18):8942-8954.
[14]WEI X B,ZHANG B,BAZEILLE N,et al. A 3'-5'exonuclease activity embedded in the helicase core domain of Candida albicans Pif1helicase[J]. Sci Rep,2017,7:42865.
[15]SABOURI N. The functions of the multi-tasking Pfh1Pif1helicase[J]. Curr Genet,2017,63(4):621-626.
[16]郭海磊,刘娜女,段晓雷,等.厌氧棒菌Pif1解旋酶的表达纯化及解旋条件优化[J].西北农林科技大学学报(自然科学版),2017,45(6):206-212.
[17]段晓雷,刘娜女,付怡欣,等.嗜热脱铁去硫弧菌Pif1解旋酶生物学活性的分子特征分析[J].中国生物化学与分子生物学报,2018,34(1):77-88.
[18]MAUNE M W,TANNER R S. Description of Anaerobaculum hydrogeniformans sp. nov.,an anaerobe that produces hydrogen from glucose,and emended description of the genus Anaerobaculum[J]. Int J Syst Evol Microbiol,2012,62(Pt 4):832-838.
[19]SANDERS C M. Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity[J]. Biochem J,010,430(1):119-128.
[20]DONATI E R,CASTRO C,URBIETA M S. Thermophilic microorganisms in biomining[J]. World J Microbiol Biotechnol,2016,32(11):179.
[21]王帅锋,刘娜女,段晓雷,等.热脱硫弧菌解旋酶基因Ty Pif1的原核表达、纯化及功能分析[J].西北农林科技大学学报(自然科学版),2016,44(9):207-212.
[22]GEORGE T,WEN Q,GRIFFITHS R,et al. Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks[J]. Nucleic Acids Res,2009,37(19):6491-6502.
[23]OSMUNDSON J S,KUMAR J,YEUNG R,et al. Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes[J]. Nat Struct Mol Biol,2017,24(2):162-170.
[24]SINGH S P,KOC K N,STODOLA J L,et al. A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End[J]. J Mol Biol,2016,428(6):1053-1067.
[25]骆衡,许厚强,陈祥,等.荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作[J].生物化学与生物物理进展,2013,40(3):255-265.
[26]PATEL D S,MISENKO S M,HER J,et al. BLM helicase regulates DNA repair by counteracting RAD51 loading at DNA double-strand break sites[J]. J Cell Biol,2017,216(11):3521-3534.