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

Journal of Molecular Biology
Saurabh P SinghRoberto Galletto

Abstract

Using a DNA polymerase coupled assay and FRET (Förster resonance energy transfer)-based helicase assays, in this work, we show that a monomer of Saccharomyces cerevisiae Pif1 can unwind dsDNA (double-stranded DNA). The helicase activity of a Pif1 monomer is modulated by the nature of the 3'-ssDNA (single-stranded 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.

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Citations

Mar 24, 2016·Nucleic Acids Research·Katrina N KocRoberto Galletto
Sep 28, 2018·The Journal of Biological Chemistry·Alicia K ByrdKevin D Raney
Nov 21, 2019·Proceedings of the National Academy of Sciences of the United States of America·Saurabh P SinghRoberto Galletto
Sep 14, 2017·Biochemical Society Transactions·Alicia K Byrd, Kevin D Raney
Nov 28, 2019·Nature Communications·Nannan SuHaiwei Song
May 18, 2016·Proceedings of the National Academy of Sciences of the United States of America·Joshua E SokoloskiTimothy M Lohman
Sep 12, 2020·The Journal of Biological Chemistry·Melanie A SparksRoberto Galletto
Jun 25, 2021·Biochemical and Biophysical Research Communications·Ke-Yu LuXu-Guang Xi
Dec 22, 2021·Biochemistry·David G Nickens, Matthew L Bochman

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