Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata

Wenlin Wu Hongyun Li Tiantian Ma Xiaobo Zhang

Wenlin Wu, Hongyun Li, Tiantian Ma, Xiaobo Zhang. Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata[J]. Acta Oceanologica Sinica. doi: 10.1007/s13131-021-1823-1
Citation: Wenlin Wu, Hongyun Li, Tiantian Ma, Xiaobo Zhang. Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata[J]. Acta Oceanologica Sinica. doi: 10.1007/s13131-021-1823-1

doi: 10.1007/s13131-021-1823-1

Characterization of DNA polymerase δ from deep-sea hydrothermal vent shrimp Rimicaris exoculata

Funds: The National Basic Research Program of China under contract No. 2015CB755903; the National Natural Science Foundation of China under contract Nos U1605214 and 31470133; the Foundation of Quanzhou Normal University under contract No. 2016YYKJ16.
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    Corresponding author: These authors contributed equally to this work.; zxb0812@zju.edu.cn
  • †These authors contributed equally to this work.
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    †These authors contributed equally to this work.
  • 1.  Characterization of vent shrimp POLD1. a. Nucleotide sequence and deduced amino acid sequence of shrimp R. exoculata POLD1. The functional domains of POLD1 were boxed. b. Phylogenetic analysis of POLD1 showing the positions of eight crustaceans used in the analysis. The tree was constructed by neighbor-joining method based on the protein sequence. The GenBank accession numbers were indicated in the parentheses. c. SDS-PAGE of expressed and purified proteins encoded by POLD1 gene of vent shrimp. Lanes M, protein molecular mass marker (kDa); 1. non-induced recombinant bacterium; 2. induced recombinant bacterium; 3. purified fusion protein His tag POLD1.

    Figure  2.  Effects of temperature on the activity and stability of POLD1. a. Optimum temperature of POLD1. DNA polymerase activity of vent shrimp POLD1 was assayed at various temperatures ranging from 15°C to 45°C. b. The thermostability of POLD1. The residual enzymatic activities were evaluated after incubation of the enzyme solution for 20 min, 40 min, 60 min or 80 min at 25°C, 30°C, 35°C, 40°C, 45°C or 50°C. In all panels, the maximum enzymatic activity was designated as 100%. Each point indicated the mean of triplicate assays within standard deviation.

    Figure  3.  Influence of pH on the DNA polymerase activity of POLD1. a. The range of pH values of vent shrimp POLD1. The DNA polymerase activity was examined at 25°C with different pH values ranging from 4.0 to 10.0. b. The DNA polymerase’s stability at different pH values. The recombinant polymerase was pre-incubated at a series of pH buffers (pH 5.0–10.0) for 20 min, 40 min, 60 min, or 80 min, followed by the DNA polymerase activity assay. The maximum enzymatic activity was designated as 100%. Each point indicated the mean of triplicate assays within standard deviation.

    Figure  4.  Effects of chelating agent, reducing agent, protease inhibitor, detergents and metal ions on the DNA polymerase activity of POLD1. a. Influence of inhibitors on the enzyme activity. The recombinant POLD1 was incubated with different inhibitors. Then the residual DNA polymerase activity was determined. The enzyme activity without inhibitors was defined as 100%. Each column indicated the mean of triplicate assays. b. Effects of metal ions on the DNA polymerase activity. The enzyme was incubated with different metal ions, followed by the examination of relative enzymatic activity. In all panels, the activity of enzyme without any treatment was designated as 100%. Each column represented the mean of triplicate within standard deviation. The significant differences between treatments and the control were indicated with asterisks (**, p<0.01).

    Figure  5.  Kinetic characterization of POLD1 using dNTP. The kinetic parameters Km and Vmax were estimated by linear regression from Lineweaver-Burk plots. All polymerase activity assays were conducted at 25°C and pH 6.5. Each point represented the mean of triplicate assays.

  • [1] Baranovskiy A G, Babayeva N D, Liston V G, et al. 2008. X-ray structure of the complex of regulatory subunits of human DNA polymerase delta. Cell Cycle, 7(19): 3026–3036. doi: 10.4161/cc.7.19.6720
    [2] Biertümpfel C, Zhao Ye, Kondo S, et al. 2010. Structure and mechanism of human DNA polymerase η. Nature, 465(7301): 1044–1048. doi: 10.1038/nature09196
    [3] Burgers P M J, Kunkel T A. 2017. Eukaryotic DNA replication fork. Annual Review of Biochemistry, 86: 417–438. doi: 10.1146/annurev-biochem-061516-044709
    [4] Desbruyères D, Biscoito M, Caprais J C, et al. 2001. Variations in deep-sea hydrothermal vent communities on the Mid-Atlantic Ridge near the Azores plateau. Deep Sea Research Part I: Oceanographic Research Papers, 48(5): 1325–1346. doi: 10.1016/S0967-0637(00)00083-2
    [5] Fortune J M, Pavlov Y I, Welch C M, et al. 2005. Saccharomyces cerevisiae DNA polymerase δ: high fidelity for base substitutions but lower fidelity for single-and multi-base deletions. Journal of Biologial Chemistry, 280(33): 29980–29987. doi: 10.1074/jbc.M505236200
    [6] Haki G D, Rakshit S K. 2003. Developments in industrially important thermostable enzymes: a review. Bioresource Technology, 89(1): 17–34. doi: 10.1016/S0960-8524(03)00033-6
    [7] Hays H, Berdis A J. 2002. Manganese substantially alters the dynamics of translesion DNA synthesis. Biochemistry, 41(15): 4771–4778. doi: 10.1021/bi0120648
    [8] Hübscher U, Maga G, Spadari S. 2002. Eukaryotic DNA polymerases. Annual Review of Biochemistry, 71: 133–163. doi: 10.1146/annurev.biochem.71.090501.150041
    [9] Hutnak M, Fisher A T, Harris R, et al. 2008. Large heat and fluid fluxes driven through mid-plate outcrops on ocean crust. Nature Geoscience, 1: 611–614. doi: 10.1038/ngeo264
    [10] Jin Y H, Obert R, Burgers P M J, et al. 2001. The 3′→5′ exonuclease of DNA polymerase δ can substitute for the 5′ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability. Proceedings of the National Academy of Sciences of the United States of America, 98(9): 5122–5127. doi: 10.1073/pnas.091095198
    [11] Johnson R E, Prakash L, Prakash S. 2012. Pol31 and Pol32 subunits of yeast DNA polymerase δ are also essential subunits of DNA polymerase ζ. Proceedings of the National Academy of Sciences of the United States of America, 109(31): 12455–12460. doi: 10.1073/pnas.1206052109
    [12] Komaï T, Segonzac M. 2008. Taxonomic review of the hydrothermal vent shrimp genera Rimicaris Williams & Rona and Chorocaris Martin & Hessler (Crustacea: Decapoda: Caridea: Alvinocarididae). Journal of Shellfish Research, 27(1): 21–41. doi: 10.2983/0730-8000(2008)27[21:TROTHV]2.0.CO;2
    [13] Konn C, Charlou J L, Holm N G, et al. 2015. The production of methane, hydrogen, and organic compounds in ultramafic-hosted hydrothermal vents of the Mid-Atlantic Ridge. Astrobiology, 15(5): 381–399. doi: 10.1089/ast.2014.1198
    [14] Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7. 0 for bigger datasets. Molecular Biology and Evolution, 33(7): 1870–1874. doi: 10.1093/molbev/msw054
    [15] Kunkel T A, Burgers P M J. 2017. Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication. Bioessays, 39(8): 1700070. doi: 10.1002/bies.201700070
    [16] Le Bloa S, Durand L, Cueff-Gauchard V, et al. 2017. Highlighting of quorum sensing lux genes and their expression in the hydrothermal vent shrimp Rimicaris exoculata ectosymbiontic community. Possible use as biogeographic markers. PLoS ONE, 12(3): e0174338
    [17] Maga G, Villani G, Tillement V, et al. 2001. Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1. Proceedings of the National Academy of Sciences of the United States of America, 98(25): 14298–14303. doi: 10.1073/pnas.251193198
    [18] Marchler-Bauer A, Derbyshire M K, Gonzales N R, et al. 2015. CDD: NCBI’s conserved domain database. Nucleic Acids Research, 43: D222–D226. doi: 10.1093/nar/gku1221
    [19] Marchler-Bauer A, Lu Shennan, Anderson J B, et al. 2011. CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Research, 39: D225–D229. doi: 10.1093/nar/gkq1189
    [20] Prindle M J, Loeb L A. 2012. DNA polymerase delta in DNA replication and genome maintenance. Environmental and Molecular Mutagenesis, 53(9): 666–682. doi: 10.1002/em.21745
    [21] Ravaux J, Cottin D, Chertemps T, et al. 2009. Hydrothermal vent shrimps display low expression of the heat-inducible hsp70 gene in nature. Marine Ecology Progress Series, 396: 153–156. doi: 10.3354/meps08293
    [22] Ravaux J, Léger N, Hamel G, et al. 2019. Assessing a species thermal tolerance through a multiparameter approach: the case study of the deep-sea hydrothermal vent shrimp Rimicaris exoculata. Cell Stress and Chaperones, 24: 647–659. doi: 10.1007/s12192-019-01003-0
    [23] Rayner E, van Gool I C, Palles C, et al. 2016. A panoply of errors: polymerase proofreading domain mutations in cancer. Nature Review Cancer, 16: 71–81. doi: 10.1038/nrc.2015.12
    [24] Seal G, Shearman C W, Loeb L A. 1979. On the fidelity of DNA replication: studies with human placenta DNA polymerases. Journal of Biological Chemistry, 254(12): 5229–5237. doi: 10.1016/S0021-9258(18)50583-4
    [25] Swan M K, Johnson R E, Prakash L, et al. 2009. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase δ. Nature Structural & Moecularl Biology, 16: 979–986
    [26] Tveit H, Kristensen T. 2001. Fluorescence-based DNA polymerase assay. Analytical Biochemistry, 289(1): 96–98. doi: 10.1006/abio.2000.4903
    [27] Vaisman A, Ling Hong, Woodgate R, et al. 2005. Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis. EMBO Journal, 24: 2957–2967. doi: 10.1038/sj.emboj.7600786
    [28] Vashishtha A K, Wang Jimin, Konigsberg W H. 2016. Different divalent cations alter the kinetics and fidelity of DNA polymerases. Journal of Biological Chemistry, 291(40): 20869–20875. doi: 10.1074/jbc.R116.742494
    [29] Vasuvat J, Montree A, Moonsom S, et al. 2016. Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ. Malaria Journal, 15: 116. doi: 10.1186/s12936-016-1166-0
    [30] Weedon M N, Ellard S, Prindle M J, et al. 2013. An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy. Nature Genetics, 45: 947–950. doi: 10.1038/ng.2670
    [31] Williams A B, Rona P A. 1986. Two new caridean shrimps (bresiliidae) from a hydrothermal field on the Mid-Atlantic Ridge. Journal of Crustacean Biology, 6(3): 446–462. doi: 10.1163/193724086X00299
    [32] Zhang Jian, Sun Qinglei, Luan Zhendong, et al. 2017. Comparative transcriptome analysis of Rimicaris sp. reveals novel molecular features associated with survival in deep-sea hydrothermal vent. Scientific Reports, 7: 2000
    [33] Zheng Ping, Wang Minxiao, Li Chaolun, et al. 2017. Insights into deep-sea adaptations and host-symbiont interactions: a comparative transcriptome study on Bathymodiolus mussels and their coastal relatives. Molecular Ecology, 26(19): 5133–5148. doi: 10.1111/mec.14160
    [34] Zhou Li, Cao Lei, Wang Xiaocheng, et al. 2020. Metal adaptation strategies of deep-sea Bathymodiolus mussels from a cold seep and three hydrothermal vents in the West Pacific. Science of the Total Environment, 707: 136046. doi: 10.1016/j.scitotenv.2019.136046
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  • 网络出版日期:  2021-06-25

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