A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3'-End.

Abstract Using a DNA polymerase coupled assay and FRET ( F orster r esonance e nergy t ransfer)-based helicase assays, in this work, we show that a monomer of Saccharomyces cerevisiae Pif1 can unwind dsDNA ( d ouble- s tranded DNA ). The helicase activity of a Pif1 monomer is modulated by the nature of the 3′-ssDNA ( s ingle- s tranded DNA ) tail of the substrate and its effect on a Pif1-dependent re-winding activity that is coupled to the opening of dsDNA. We propose that, in addition to the ssDNA site on the protein that interacts with the translocating strand, Pif1 has a second site that binds the 3′-ssDNA of the substrate. Interaction of DNA with this site modulates the degree to which re-winding counteracts unwinding. Depending on the nature of the 3′-tail and the length of the duplex DNA to be unwound, this activity is sufficiently strong to mask the helicase activity of a monomer. In excess Pif1 over the DNA, the Pif1-dependent re-winding of the opened DNA strongly limits unwinding, independent of the 3′-tail. We propose that, in this case, binding of DNA to the second site is precluded and modulation of the Pif1-dependent re-winding activity is largely lost.