The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium Thermotoga elfii is an Accessory Single-Stranded DNA Binding Module

Biochemistry. 2018 Feb 20;57(7):1108-1118. doi: 10.1021/acs.biochem.7b01233. Epub 2018 Jan 30.

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 homologues. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • DNA Helicases / chemistry
  • DNA Helicases / metabolism*
  • DNA, Single-Stranded / metabolism*
  • Gram-Negative Anaerobic Straight, Curved, and Helical Rods / chemistry
  • Gram-Negative Anaerobic Straight, Curved, and Helical Rods / metabolism*
  • Protein Binding
  • Protein Domains
  • Sequence Alignment

Substances

  • Bacterial Proteins
  • DNA, Single-Stranded
  • Adenosine Triphosphatases
  • DNA Helicases