Positioning the 5-flap junction in the active site controls the rate of flap endonuclease-1- catalyzed DNA cleavage

Bo Song, Samir M. Hamdan, Manju M. Hingorani*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2 have suggested that base unpairing at the 5-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg2. We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5-flap:template junction, indicating that local DNA distortion (e.g. disruption of base stacking observed in structures), associated with positioning the 5-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5 to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5-flaps.

Original languageEnglish (US)
Pages (from-to)4792-4804
Number of pages13
JournalJournal of Biological Chemistry
Issue number13
StatePublished - Mar 30 2018

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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