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

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.

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

“…Thus, the chase experiment reveals a two-step process of product release: first, rapid isomerization of the nicked DNA product after cleavage (perhaps partial relaxation of the bent state along with release of the 5Ј-flap product), followed by slower dissociation from hFEN1 (and full unbending) to complete the catalytic turnover. Recent single-molecule measurements also indicate a similar two-step product release mechanism (29). This conformational change and slow release process could facilitate coordinated handoff of the nicked duplex from hFEN1 to DNA ligase.…”

“…2 and Table 1). We recently observed these two steps of nicked product release by single-molecule imaging as well (29). Such a prolonged dwell time could enable direct handoff of the nicked duplex from hFEN1 to DNA ligase, thus minimizing release of a genome-destabilizing intermediate.…”

Structure of the processive human Pol δ holoenzyme

“…However, handing off a bent DNA is not a must for FEN1 activity since FEN1 can actively bend the nick junction in diffusion-limited kinetics 54,55 . Nick translation occurs at rates that are 10-fold faster than nick DNA product release by FEN1 16,[55][56][57] . This suggests that Pol δ and FEN1 must be actively handing off their products during nick translation.…”

“…It is unclear how Pol δ-PCNA-FEN1 would signal the binding of Lig1. Interestingly, FEN1 releases the nick product in two steps, where it binds it briefly in a bent conformer that is followed by a lengthy binding in an extended conformer 55 . It is possible that the bent conformer is more compatible with Pol δ binding while the extended one sequesters the nick until Pol δ moves out of the way Interacting phosphates on the template and primer strands are shown in red and yellow, respectively.…”

Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

“…These longer flaps are coated by replication protein A, which inhibits Fen1/Rad27 nuclease activity but promotes Dna2 nuclease activity, which shortens the flaps [153][154][155]. After shortening, Fen1/Rad27 processes the final step to generate a nick, which is sealed by DNA ligase I [156]. A role for ScPif1 in Okazaki maturation has been proposed, as disruption of ScPif1 nuclear function, but not RRM3 deletion, suppresses defects caused by dna2-1 and dna2-2 mutations as well as negative genetic interactions of DNA2, but not those with genes that have roles in Okazaki fragment processing [48].…”