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Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase–helicase from Arabidopsis thaliana

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Abstract

An open reading frame from Arabidopsis thaliana, which is highly homologous to the human mitochondrial DNA helicase TWINKLE, was previously cloned, expressed, and shown to have DNA primase and DNA helicase activity. The level of DNA primase activity of this Arabidopsis Twinkle homolog (ATH) was low, perhaps due to an incomplete zinc binding domain (ZBD). In this study, N-terminal truncations of ATH implicate residues 80–102 interact with the RNA polymerase domain (RPD). In addition, chimeric proteins, constructed using domains from ATH and the well-characterized T7 phage DNA primase–helicase gp4, were created to determine if the weak primase activity of ATH could be enhanced. Two chimeric proteins were constructed: ATHT7 contains the ZBD and RPD domains of ATH tethered to the helicase domain of T7, while T7ATH contains the ZBD and RPD domains of T7 tethered to the helicase domain of ATH. Both chimeric proteins were successfully expressed and purified in E. coli, and assayed for traditional primase and helicase activities. T7ATH was able to generate short oligoribonucleotide primers, but these primers could not be cooperatively extended by a DNA polymerase. Although T7ATH contains the ATH helicase domain, it exhibited few of the characteristics of a functional helicase. ATHT7 lacked primase activity altogether and also demonstrated only weak helicase activities. This work demonstrates the importance of interactions between structurally and functionally distinct domains, especially in recombinant, chimeric proteins.

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Acknowledgments

This work was supported by National Institutes of Health [Grant Number R15-GM086838-01A1, awarded to DJC]. We are grateful to Tatenda Kadungure, and Abhijit Srungavarapu for exceptional technical assistance.

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Author notes

  1. Jamie B. Towle-Weicksel

    Present address: Department of Therapeutic Radiology, Yale University, New Haven, CT, USA

  2. Yun Cao

    Present address: WuXi App Tech, Shanghai, China

  3. Lisa J. Crislip

    Present address: Center for Cutaneous Oncology, Dana Farber Cancer Institute, Boston, MA, USA

  4. Donald J. Crampton

    Present address: Mills College, Oakland, CA, USA

Authors and Affiliations

  1. Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA, 01610, USA

    Jamie B. Towle-Weicksel, Yun Cao, Lisa J. Crislip, David L. Thurlow & Donald J. Crampton

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  1. Jamie B. Towle-Weicksel
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Correspondence to Jamie B. Towle-Weicksel.

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Towle-Weicksel, J.B., Cao, Y., Crislip, L.J. et al. Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase–helicase from Arabidopsis thaliana . Mol Biol Rep 41, 7783–7795 (2014). https://doi.org/10.1007/s11033-014-3671-y

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