Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

J Biol Chem. 2008 Oct 10;283(41):27483-27493. doi: 10.1074/jbc.M804550200. Epub 2008 Aug 9.


Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetyltransferases
  • DNA / genetics
  • DNA / metabolism*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism
  • DNA Replication / physiology*
  • DNA, Fungal / biosynthesis*
  • DNA, Fungal / genetics
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Oligoribonucleotides / genetics
  • Oligoribonucleotides / metabolism*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*


  • DNA, Fungal
  • Membrane Proteins
  • Okazaki fragments
  • Oligoribonucleotides
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Acetyltransferases
  • ELO2 protein, S cerevisiae
  • DNA Polymerase III
  • PIF1 protein, S cerevisiae
  • DNA Helicases
  • DNA2 protein, S cerevisiae