Title
The Pif1 signature motif of Pfh1 is necessary for both protein displacement and helicase unwinding activities, but is dispensable for strand-annealing activity
Author
Jani B. Mohammad, Marcus Wallgren, Nasim Sabouri
Year
2018
Journal
Nucleic Acids Research
Abstract
Pfh1, the sole member of the Pif1 helicases in Schizosaccharomyces pombe, is multifunctional and essential for maintenance of both the nuclear and mitochondrial genomes. However, we lack mechanistic insights into the functions of Pfh1 and its different motifs. This paper is specifically concerned with the importance of the Pif1 signature motif (SM), a 23 amino acids motif unique to Pif1 helicases, because a single amino acid substitution in this motif is associated with increased risk of breast cancer in humans and inviability in S. pombe. Here we show that the nuclear isoform of Pfh1 (nPfh1) unwound RNA/DNA hybrids more efficiently than DNA/DNA, suggesting that Pfh1 resolves RNA/DNA structures like R-loops in vivo. In addition, nPfh1 displaced proteins from DNA and possessed strand-annealing activity. The unwinding and protein displacement activities were dependent on the SM because nPfh1 without a large portion of this motif (nPfh1-21) or with the disease/inviability-linked mutation (nPfh1- L430P) lost these properties. Unexpectedly, both nPfh1-L430P and nPfh1-21 still displayed binding to G-quadruplex DNA and demonstrated strandannealing activity. Misregulated strand annealing and binding of nPfh1-L430P without unwinding are perhaps the reasons that cells expressing this allele are inviable.
Instrument
J-710
Keywords
Circular dichroism, Secondary structure, Biochemistry