Sulfolobus acidocaldarius DNA聚合酶Ⅳ跨越损伤合成能力的酶学特征
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摘要
【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DNA聚合酶Ⅳ (Saci_0554)为例,表征其跨越模板上损伤碱基的DNA合成效果。【方法】将DNA聚合酶Ⅳ (SacpolⅣ)在大肠杆菌中进行重组表达,经亲和层析纯化得到SacpolⅣ蛋白;利用人工合成的带有不同损伤的寡核苷酸片段作为模板DNA,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定SacpolⅣ在体外跨越各种损伤碱基进行跨损伤合成的催化能力。【结果】SacpolⅣ重组蛋白能够不同程度地跨越嘌呤和嘧啶损伤,跨越能力的高低取决于损伤碱基与正常碱基形成氢键的能力。本研究还发现,SacpolⅣ能够在DNA链中掺入核糖核苷酸,但掺入核糖核苷酸的效率低于脱氧核糖核苷酸。【结论】本研究证实SacpolⅣ具有很强的跨越损伤合成能力,能够跨越多种氢键配对能力减弱的损伤碱基,为其在细胞内的跨越损伤合成功能提供了生化证据。
[Objective] To characterize the translesion synthesis of different damages by DNA polymerase Ⅳ from thermophilic archaea Sulfolobus acidocaldarius. [Methods] We detected the translesion synthesis ability of Sulfolobus acidocaldarius polymerase Ⅳ(SacpolⅣ) using fluorescence labeled oligonucleotides as substrates through denaturing polyacrylamide gel electrophoresis. [Results] We successfully purified SacpolⅣ from induced E. coli and confirmed SacpolⅣ possessed strong translesion synthesis ability to DNA damages on purines and pyrimidines. The TLS ability depends on whether damaged bases can pair normal bases perfectly. Besides, we found Sacpol Ⅳ can incorporate rNMP into DNA strand. [Conclusion] We confirmed that SacpolⅣ is a typical TLS DNA polymerase with the abilities of bypassing damaged purines and pyrimidines on DNA, and incorporating rNMP into DNA.
[Objective] To characterize the translesion synthesis of different damages by DNA polymerase Ⅳ from thermophilic archaea Sulfolobus acidocaldarius. [Methods] We detected the translesion synthesis ability of Sulfolobus acidocaldarius polymerase Ⅳ(SacpolⅣ) using fluorescence labeled oligonucleotides as substrates through denaturing polyacrylamide gel electrophoresis. [Results] We successfully purified SacpolⅣ from induced E. coli and confirmed SacpolⅣ possessed strong translesion synthesis ability to DNA damages on purines and pyrimidines. The TLS ability depends on whether damaged bases can pair normal bases perfectly. Besides, we found Sacpol Ⅳ can incorporate rNMP into DNA strand. [Conclusion] We confirmed that SacpolⅣ is a typical TLS DNA polymerase with the abilities of bypassing damaged purines and pyrimidines on DNA, and incorporating rNMP into DNA.
引文
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[24]Zahn KE,Wallace SS,DoubliéS.DNA polymerases provide a canon of strategies for translesion synthesis past oxidatively generated lesions.Current Opinion in Structural Biology,2011,21(3):358-369.
[25]Kirouac KN,Ling H.Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNApolymerase iota.Proceedings of the National Academy of Sciences of the United States of America,2011,108(8):3210-3215.
[26]Lindahl T,Nyberg B.Rate of depurination of native deoxyribonucleic acid.Biochemistry,1972,11(19):3610-3618.
[27]Kokoska RJ,McCulloch SD,Kunkel TA.The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymeraseηand Sulfolobus solfataricus Dpo4.Journal of Biological Chemistry,2003,278(50):50537-50545.
[28]Brock TD,Brock KM,Belly RT,Weiss RL.Sulfolobus:a new genus of sulfur-oxidizing bacteria living at low pH and high temperature.Archiv für Mikrobiologie,1972,84(1):54-68.
[29]Chen LM,Brügger K,Skovgaard M,Redder P,She QX,Torarinsson E,Greve B,Awayez M,Zibat A,Klenk HP,Garrett RA.The genome of Sulfolobus acidocaldarius,a model organism of the Crenarchaeota.Journal of Bacteriology,2005,187(14):4992-4999.
[30]Wagner M,van Wolferen M,Wagner A,Lassak K,Meyer BH,Reimann J,Albers SV.Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius.Frontiers in Microbiology,2012,3:214.
[31]Peng L,Xia X,Liu XP.Biochemical characterization of translesion synthesis by Sulfolobus acidocaldarius DNApolymerases.Chemical Research in Chinese Universities,2016,32(2):226-233.
[32]Lindahl T,Nyberg B.Heat-induced deamination of cytosine residues in deoxyribonucleic acid.Biochemistry,1974,13(16):3405-3410.
[33]Cadet J,Berger M,Douki T,Ravanat JL.Oxidative damage to DNA:formation,measurement,and biological significance.Reviews of Physiology,Biochemistry and Pharmacology,1997,131:1-87.
[34]Bebenek K,Garcia-Diaz M,Patishall SR,Kunkel TA.Biochemical properties of Saccharomyces cerevisiae DNApolymerase IV.Journal of Biological Chemistry,2005,280(20):20051-20058.
[35]Zhang LK,Lou HQ,Guo L,Zhan ZY,Duan ZH,Guo X,Huang L.Accurate DNA synthesis by Sulfolobus solfataricus DNA polymerase B1 at high temperature.Extremophiles,2010,14:107.
[36]Boudsocq F,Kokoska RJ,Plosky BS,Vaisman A,Ling H,Kunkel TA,Yang W,Woodgate R.Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication.Journal of Biological Chemistry,2004,279(31):32932-32940.
[2]Lindahl T,Barnes DE.Repair of endogenous DNA damage.Cold Spring Harbor Symposia on Quantitative Biology,2000,65:127-134.
[3]Ohmori H,Friedberg EC,Fuchs RPP,Goodman MF,Hanaoka F,Hinkle D,Kunkel TA,Lawrence CW,Livneh Z,Nohmi T,Prakash L,Prakash S,Todo T,Walker GC,Wang ZG,Woodgate R.The Y-family of DNA polymerases.Molecular Cell,2001,8(1):7-8.
[4]Sale JE,Lehmann AR,Woodgate R.Y-family DNApolymerases and their role in tolerance of cellular DNAdamage.Nature Reviews Molecular Cell Biology,2012,13(3):141-152.
[5]Yang W,Woodgate R.What a difference a decade makes:insights into translesion DNA synthesis.Proceedings of the National Academy of Sciences of the United States of America,2007,104(40):15591-15598.
[6]Muniandy PA,Liu J,Majumdar A,Liu ST,Seidman MM.DNA interstrand crosslink repair in mammalian cells:step by step.Critical Reviews in Biochemistry and Molecular Biology,2010,45(1):23-49.
[7]Sale JE.Competition,collaboration and coordination-determining how cells bypass DNA damage.Journal of Cell Science,2012,125(7):1633-1643.
[8]Andersen PL,Xu F,Xiao W.Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA.Cell Research,2008,18(1):162-173.
[9]Lehmann AR,Niimi A,Ogi T,Brown S,Sabbioneda S,Wing JF,Kannouche PL,Green CM.Translesion synthesis:Y-family polymerases and the polymerase switch.DNARepair,2007,6(7):891-899.
[10]Izhar L,Ziv O,Cohen IS,Geacintov NE,Livneh Z.Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells.Proceedings of the National Academy of Sciences of the United States of America,2013,110(16):E1462-E1469.
[11]Pata JD.Structural diversity of the Y-family DNApolymerases.Biochimica et Biophysica Acta(BBA)-Proteins and Proteomics,2010,1804(5):1124-1135.
[12]Jarosz DF,Godoy VG,Walker GC.Proficient and accurate bypass of persistent DNA lesions by DinB DNA polymerases.Cell Cycle,2007,6(7):817-822.
[13]Yang W.An overview of Y-family DNA polymerases and a case study of human DNA polymeraseη.Biochemistry,2014,53(17):2793-2803.
[14]Johnson RE,Kondratick CM,Prakash S,Prakash L.hRAD30mutations in the variant form of xeroderma pigmentosum.Science,1999,285(5425):263-265.
[15]Masutani C,Kusumoto R,Yamada A,Dohmae N,Yokoi M,Yuasa M,Araki M,Iwai S,Takio KJ,Hanaoka F.The XPV(xeroderma pigmentosum variant)gene encodes human DNApolymeraseη.Nature,1999,399(6737):700-704.
[16]McDonald JP,Frank EG,Plosky BS,Rogozin IB,Masutani C,Hanaoka F,Woodgate R,Gearhart PJ.129-derived strains of mice are deficient in DNA polymeraseιand have normal immunoglobulin hypermutation.Journal of Experimental Medicine,2003,198(4):635-643.
[17]Xu PN,Oum L,Geacintov NE,Broyde S.Nucleotide selectivity opposite a benzo[a]pyrene-derived N2-dG adduct in a Y-family DNA polymerase:a 5?-slippage mechanism.Biochemistry,2008,47(9):2701-2709.
[18]Ling H,Sayer JM,Plosky BS,Yagi H,Boudsocq F,Woodgate R,Jerina DM,Yang W.Crystal structure of a benzo[a]pyrene diol epoxide adduct in a ternary complex with a DNApolymerase.Proceedings of the National Academy of Sciences of the United States of America,2004,101(8):2265-2269.
[19]Walsh JM,Ippoliti PJ,Ronayne EA,Rozners E,Beuning PJ.Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily.DNA Repair,2013,12(9):713-722.
[20]Zhou Y,Wang JL,Zhang YB,Wang ZG.The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.Nucleic Acids Research,2010,38(15):5036-5046.
[21]Guo CX,Fischhaber PL,Luk-Paszyc MJ,Masuda Y,Zhou J,Kamiya K,Kisker C,Friedberg EC.Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis.The EMBO Journal,2003,22(24):6621-6630.
[22]Maga G,Villani G,Crespan E,Wimmer U,Ferrari E,Bertocci B,Hübscher U.8-oxo-guanine bypass by human DNApolymerases in the presence of auxiliary proteins.Nature,2007,447(7144):606-608.
[23]Haracska L,Yu SL,Johnson RE,Prakash L,Prakash S.Efficient and accurate replication in the presence of7,8-dihydro-8-oxoguanine by DNA polymeraseη.Nature Genetics,2000,25(4):458-461.
[24]Zahn KE,Wallace SS,DoubliéS.DNA polymerases provide a canon of strategies for translesion synthesis past oxidatively generated lesions.Current Opinion in Structural Biology,2011,21(3):358-369.
[25]Kirouac KN,Ling H.Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNApolymerase iota.Proceedings of the National Academy of Sciences of the United States of America,2011,108(8):3210-3215.
[26]Lindahl T,Nyberg B.Rate of depurination of native deoxyribonucleic acid.Biochemistry,1972,11(19):3610-3618.
[27]Kokoska RJ,McCulloch SD,Kunkel TA.The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymeraseηand Sulfolobus solfataricus Dpo4.Journal of Biological Chemistry,2003,278(50):50537-50545.
[28]Brock TD,Brock KM,Belly RT,Weiss RL.Sulfolobus:a new genus of sulfur-oxidizing bacteria living at low pH and high temperature.Archiv für Mikrobiologie,1972,84(1):54-68.
[29]Chen LM,Brügger K,Skovgaard M,Redder P,She QX,Torarinsson E,Greve B,Awayez M,Zibat A,Klenk HP,Garrett RA.The genome of Sulfolobus acidocaldarius,a model organism of the Crenarchaeota.Journal of Bacteriology,2005,187(14):4992-4999.
[30]Wagner M,van Wolferen M,Wagner A,Lassak K,Meyer BH,Reimann J,Albers SV.Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius.Frontiers in Microbiology,2012,3:214.
[31]Peng L,Xia X,Liu XP.Biochemical characterization of translesion synthesis by Sulfolobus acidocaldarius DNApolymerases.Chemical Research in Chinese Universities,2016,32(2):226-233.
[32]Lindahl T,Nyberg B.Heat-induced deamination of cytosine residues in deoxyribonucleic acid.Biochemistry,1974,13(16):3405-3410.
[33]Cadet J,Berger M,Douki T,Ravanat JL.Oxidative damage to DNA:formation,measurement,and biological significance.Reviews of Physiology,Biochemistry and Pharmacology,1997,131:1-87.
[34]Bebenek K,Garcia-Diaz M,Patishall SR,Kunkel TA.Biochemical properties of Saccharomyces cerevisiae DNApolymerase IV.Journal of Biological Chemistry,2005,280(20):20051-20058.
[35]Zhang LK,Lou HQ,Guo L,Zhan ZY,Duan ZH,Guo X,Huang L.Accurate DNA synthesis by Sulfolobus solfataricus DNA polymerase B1 at high temperature.Extremophiles,2010,14:107.
[36]Boudsocq F,Kokoska RJ,Plosky BS,Vaisman A,Ling H,Kunkel TA,Yang W,Woodgate R.Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication.Journal of Biological Chemistry,2004,279(31):32932-32940.