Heterologous expression and characterization of unwinding reaction of Pif1 helicase from Anaerobaculum hydrogeniforman
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    Abstract:

    [Objective] We expressed and purified a Pif1 helicase from Anaerobaculum hydrogeniforman to assess its unwinding characteristics.[Methods] Recombinant vector pET21a-Ana.Pif1-TEV/SUMO was constructed and expressed in E. coli BL21(DE3), and Ana.Pif1 protein was purified using Ni-NTAs and Superdex 200 column chromatography. We then evaluated the unwinding polarity, optimal ATP concentration, optimal metal cofactor, optimal unwinding temperature, and substrate specificity of Ana.Pif1 by stopped-flow FRET assay. [Results] The molecular weight of Ana.Pif1 helicase was 59 kDa with no tags, protein yield was 9.5 mg/L, and purity was about 97%. We confirmed the unwinding polarity of Ana.Pif1 was from DNA 5' to 3' end. And we found the optimal ATP concentration for Ana.Pif1 unwinding to be 2 mmol/L, the optimal metal cofactor to be Mg2+, and the optimal reaction temperature to be 55℃. The substrate specificity of Ana.Pif1 unwinding DNA replication intermediates indicated that the Y-S fork substrate had the highest unwinding rate, 0.127 s-1, and 12 nt-bubble substrate had the largest unwinding amplitude, 78.8%, indicating these two substrates could be natural replication intermediates for Ana.Pif1. [Conclusion] This study is the first to systematically analyze the unwinding characteristics of Ana.Pif1 helicase. These findings facilitate elucidation of the molecular mechanisms of these thermophilic bacteria Pif1-family helicases.

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Xiaolei Duan, Nan&#; Liu, Tao Zhang, Rui Cao, Junyao Jiang, Xun Min, Xuguang Xi. Heterologous expression and characterization of unwinding reaction of Pif1 helicase from Anaerobaculum hydrogeniforman. [J]. Acta Microbiologica Sinica, 2019, 59(3): 566-577

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  • Received:May 11,2018
  • Revised:July 13,2018
  • Online: March 01,2019
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