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The WYL domain of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii is an accessory single-stranded DNA binding module

Nicholas M. Andis, Christopher W. Sausen, Ashna Alladin, Matthew L. Bochman
doi: https://doi.org/10.1101/163188
Nicholas M. Andis
†Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405, USA
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Christopher W. Sausen
†Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405, USA
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Ashna Alladin
†Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405, USA
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Matthew L. Bochman
†Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405, USA
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ABSTRACT

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologs. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.

  • ABBREVIATIONS

    dsDNA
    double-stranded DNA
    ssDNA
    single-stranded DNA
    hPIF1
    human PIF1
    TePif1
    Pseudothermotoga elfii Pif1
    ToPif1
    Thermus oshimai Pif1
    TyPif1
    Thermodesulfovibrio yellowstonii Pif1
    HDA
    helicase dependent amplification
    MW
    molecular weight
    TePif1ΔWYL
    TePif1 lacking its C-terminal WYL domain
    TePif1-4x
    TePif1 containing R470A, C494A, R501A, and R504A mutations
    CV
    column volume
    TLC
    thin-layer chromatography
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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    Posted January 16, 2018.
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    The WYL domain of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii is an accessory single-stranded DNA binding module
    Nicholas M. Andis, Christopher W. Sausen, Ashna Alladin, Matthew L. Bochman
    bioRxiv 163188; doi: https://doi.org/10.1101/163188
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    The WYL domain of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii is an accessory single-stranded DNA binding module
    Nicholas M. Andis, Christopher W. Sausen, Ashna Alladin, Matthew L. Bochman
    bioRxiv 163188; doi: https://doi.org/10.1101/163188

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