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Published October 10, 2008 | Published
Journal Article Open

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


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 Pol 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.

Additional Information

© 2008 the American Society for Biochemistry and Molecular Biology. Submitted on June 13, 2008. Revised on August 1, 2008. Accepted on August 9, 2008. Papers In Press, published online ahead of print August 9, 2008. J. Biol. Chem, 10.1074/jbc.M804550200. We thank the members of the Bambara laboratory for helpful discussions and suggestions. This work was supported by National Institutes of Health grant GM024441 (to R.A.B.) with additional support from NIH grants GM087666 (to J.L.C.), GM32431 (to P.M.J.B.), and GM068411 (to J.E.P.).

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