Single-molecule visualization of Pif1 helicase translocation on single-stranded DNA

Mainak Mustafi, Youngho Kwon, Patrick Sung, Eric C. Greene

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Pif1 is a broadly conserved helicase that is essential for genome integrity and participates in numerous aspects of DNA metabolism, including telomere length regulation, Okazaki fragment maturation, replication fork progression through difficult-to-replicate sites, replication fork convergence, and break-induced replication. However, details of its translocation properties and the importance of amino acids residues implicated in DNA binding remain unclear. Here, we use total internal reflection fluorescence microscopy with single-molecule DNA curtain assays to directly observe the movement of fluorescently tagged Saccharomyces cerevisiae Pif1 on single-stranded DNA (ssDNA) substrates. We find that Pif1 binds tightly to ssDNA and translocates very rapidly (∼350 nucleotides per second) in the 5’→3′ direction over relatively long distances (∼29,500 nucleotides). Surprisingly, we show the ssDNA–binding protein replication protein A inhibits Pif1 activity in both bulk biochemical and single-molecule measurements. However, we demonstrate Pif1 can strip replication protein A from ssDNA, allowing subsequent molecules of Pif1 to translocate unimpeded. We also assess the functional attributes of several Pif1 mutations predicted to impair contact with the ssDNA substrate. Taken together, our findings highlight the functional importance of these amino acid residues in coordinating the movement of Pif1 along ssDNA.

Original languageEnglish (US)
Article number104817
JournalJournal of Biological Chemistry
Volume299
Issue number6
DOIs
StatePublished - Jun 2023

Keywords

  • DNA curtains
  • Pif1 helicase
  • Pif1 processivity
  • SF1B helicase
  • single-molecule fluorescence
  • ssDNA binding
  • ssDNA translocation

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology

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