Sulfolobus acidocaldarius DHH超家族核酸酶Saci0542的酶学特征研究
作者:
基金项目:

国家重点研究发展计划(2017YFA0504901)


Enzymatic characterization of Sulfolobus acidocaldarius Saci0542 nuclease belonging to DHH superfamily
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [25]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DHH超家族核酸酶(Saci0542)为例,研究其核酸外切酶活性特点,为阐明其在DNA代谢中的具体功能提供生化基础。【方法】将嗜酸嗜热硫化叶菌DHH超家族核酸酶Saci0542基因在大肠杆菌中重组表达,经亲和层析纯化得到电泳纯的重组蛋白;利用荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定Saci0542的酶学特征。【结果】重组表达的DHH超家族核酸酶Saci0542具有典型的单链核酸特异性的3'-5'外切酶活性。进一步酶学特征表征结果如下:酶活性依赖于二价金属离子Mn2+,而Ca2+、Mg2+、Zn2+等二价金属离子对活性没有明显的促进作用;Saci0542在pH 5.5–10的广泛范围内均表现出较高酶活性;高于200 mmol/L的NaCl强烈抑制酶活性;最适反应温度为50–55℃;末端磷酸基团抑制3'-5'外切酶活性。【结论】本研究证实,Saci0542是一种Mn2+依赖型3'-5'外切酶,酶活性与NrnA核酸酶相似,可能在细胞内负责DNA修复或RNA的降解再循环利用。

    Abstract:

    [Objective] To provide biochemical support for studying the potential function during DNA repair via systematically characterizing the enzymatic properties of the DHH superfamily nuclease (Saci0542) of Sulfolobus acidocaldarius. [Methods] Saci0542 was recombinantly expressed in Escherichia coli and then purified by affinity chromatography. The enzymatic properties of Saci0542 were characterized in vitro with fluorescence-labeled oligonucleotides as substrates by urea-denaturing polyacrylamide gel electrophoresis. [Results] The recombinant Saci0542 had a typical 3'-5' exonuclease activity on ssDNA. Its activity was dependent on the divalent metal ion Mn2+ and inhibited by the divalent metal ions such as Ca2+, Mg2+, and Zn2+. Saci0542 showed high activity in a wide range of pH 5.5-10. NaCl above 50 mmol/L strongly inhibited the activity of this enzyme, and the optimum reaction temperature was 50-55℃. The terminal phosphate group inhibited the 3'-5' exonuclease activity. [Conclusion] This study confirmed that Saci0542 was a Mn2+-dependent 3'-5' exonuclease with the activity similar to that of NrnA nuclease, which may be responsible for DNA repair or RNA degradation and recycling in the cell.

    参考文献
    [1] Makarova KS, Koonin EV, Kelman Z. The CMG (CDC45/RecJ, MCM, GINS) complex is a conserved component of the DNA replication system in all archaea and eukaryotes. Biology Direct, 2012, 7(1):1-10.
    [2] Fabrichniy IP, Lehtiö L, Tammenkoski M, Zyryanov AB, Oksanen E, Baykov AA, Lahti R, Goldman A. A trimetal site and substrate distortion in a family II inorganic pyrophosphatase. Journal of Biological Chemistry, 2007, 282(2):1422-1431.
    [3] Goldman SR, Sharp JS, Vvedenskaya IO, Livny J, Dove SL, Nickels BE. NanoRNAs prime transcription initiation in vivo. Molecular Cell, 2011, 42(6):817-825.
    [4] Nickels BE, Dove SL. NanoRNAs:a class of small RNAs that can prime transcription initiation in bacteria. Journal of Molecular Biology, 2011, 412(5):772-781.
    [5] Yu D, Deutscher MP. Oligoribonuclease is distinct from the other known exoribonucleases of Escherichia coli. Journal of Bacteriology, 1995, 177(14):4137-4139.
    [6] Ghosh S, Deutscher MP. Oligoribonuclease is an essential component of the mRNA decay pathway. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(8):4372-4377.
    [7] Zuo Y, Deutscher MP. Exoribonuclease superfamilies:structural analysis and phylogenetic distribution. Nucleic Acids Research, 2001, 29(5):1017-1026.
    [8] Niyogi SK, Datta AK. A novel oligoribonuclease of Escherichia coli. I. Isolation and properties. Journal of Biological Chemistry, 1975, 250(18):7307-7312.
    [9] Datta AK, Niyogi K. A novel oligoribonuclease of Escherichia coli. II. Mechanism of action. Journal of Biological Chemistry, 1975, 250(18):7313-7319.
    [10] Mechold U, Ogryzko V, Ngo S, Danchin A. Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells. Nucleic Acids Research, 2006, 34(8):2364-2373.
    [11] Danchin A. A phylogenetic view of bacterial ribonucleases. Progress in Molecular Biology and Translational Science, 2009, 85:1-41.
    [12] Mechold U, Fang G, Ngo S, Ogryzko V, Danchin A. YtqI from Bacillus subtilis has both oligoribonuclease and pAp-phosphatase activity. Nucleic Acids Research, 2007, 35(13):4552-4561.
    [13] Condon C, Pellegrini O, Mathy N, Bénard L, Redko Y, Oussenko I A, Gintaras D, Bechhofer DH. Assay of Bacillus subtilis ribonucleases in vitro. Methods in Enzymology, 2008, 447:277-308.
    [14] Uemura Y, Nakagawa N, Wakamatsu T, Kim, K, Montelione GT, Hunt JF, Kuramitsu S, Masui R. Crystal structure of the ligand-binding form of nanoRNase from Bacteroides fragilis, a member of the DHH/DHHA1 phosphoesterase family of proteins. FEBS Letters, 2013, 587(16):2669-2674.
    [15] Srivastav R, Kumar D, Grover A, Singh A, Manjasetty BA, Sharma R, Taneja B. Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases. Nucleic Acids Research, 2014, 42(12):7894-7910.
    [16] Schmier BJ, Nelersa CM, Malhotra A. Structural basis for the bidirectional activity of Bacillus nanoRNase NrnA. Scientific Reports, 2017, 7(1):1-13.
    [17] Fang M, Zeisberg WM, Condon C, Ogryzko V, Danchin A, Mechold U. Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis. Nucleic Acids Research, 2009, 37(15):5114-5125.
    [18] Liu MF, Cescau S, Mechold U, Wang J, Cohen D, Danchin A, Boulouis HJ, Biville F. Identification of a novel nanoRNase in Bartonella. Microbiology, 2012, 158(4):886-895.
    [19] Deng YJ, Feng L, Zhou H, Xiao X, Wang FP, Liu XP. NanoRNase from Aeropyrum pernix shows nuclease activity on ssDNA and ssRNA. DNA Repair, 2018, 65:54-63.
    [20] 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.
    [21] Chen L, Brügger K, Skovgaard M, Redder P, She Q, Torarinsson E, Garrett RA. The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota. Journal of Bacteriology, 2005, 187(14):4992-4999.
    [22] 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.
    [23] Yi GS, Song Y, Wang WW, Chen JN, Deng W, Cao WG, Wang FP, Xiao X, Liu, XP. Two archaeal RecJ nucleases from Methanocaldococcus jannaschii show reverse hydrolysis polarity:implication to their unique function in archaea. Genes, 2017, 8(9):211.
    [24] Ogino H, Ishino S, Kohda D, Ishino Y. The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3'-5' exonuclease that associates with a DNA replication complex. Journal of Biological Chemistry, 2017, 292(19):7921-7931.
    [25] Huang J, Lu J, Barany F, Cao W. Multiple cleavage activities of endonuclease V from Thermotoga maritima:recognition and strand nicking mechanism. Biochemistry, 2001, 40(30):8738-8748.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

王伟玮,谢娟娟,宋吴涛,刘喜朋,汪启胜. Sulfolobus acidocaldarius DHH超家族核酸酶Saci0542的酶学特征研究[J]. 微生物学报, 2022, 62(2): 556-566

复制
分享
文章指标
  • 点击次数:377
  • 下载次数: 1202
  • HTML阅读次数: 925
  • 引用次数: 0
历史
  • 收稿日期:2021-04-22
  • 最后修改日期:2021-08-14
  • 在线发布日期: 2022-01-28
文章二维码