Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

Nucleic Acids Res. 2018 Apr 6;46(6):2956-2974. doi: 10.1093/nar/gky082.

Abstract

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyltransferases / genetics*
  • Acetyltransferases / metabolism
  • DNA / genetics*
  • DNA Cleavage*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism
  • DNA Replication / genetics*
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism
  • Fluorescence Resonance Energy Transfer / methods
  • Kinetics
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Protein Binding
  • Replication Protein A / genetics
  • Replication Protein A / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction / genetics
  • Single Molecule Imaging / methods
  • Substrate Specificity

Substances

  • DNA, Fungal
  • Membrane Proteins
  • Okazaki fragments
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Acetyltransferases
  • ELO2 protein, S cerevisiae
  • DNA Helicases
  • DNA2 protein, S cerevisiae