Abstract
Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.
Highlights
The activity of Flap Endonuclease 1 (Fen1) as a divalent metal ion-dependent phosphodiesterase is essential to maintain genome integrity in all domains of life [1,2]
The double-flap substrate contains two duplex regions that we have termed the 50-flap-duplex (5F-duplex which refers to the duplex containing the 50-flap strand) and the 30-flap-duplex (3F-duplex, which refers to the duplex region containing the 30-flap strand, see Figure 1)
It has been suggested that Fen1 specificity for certain flap substrates may be linked to their intrinsic flexibility and potential to become distorted by Fen1 [1,2,3]
Summary
The activity of Flap Endonuclease 1 (Fen1) as a divalent metal ion-dependent phosphodiesterase is essential to maintain genome integrity in all domains of life [1,2]. In DNA repair processes, Fen is required for non-homologous end joining of double-stranded DNA breaks and for long-patch base-excision repair (lpBER) [1,2,7]. Consistent with this critical role of Fen preventing genome instability, mutations that decrease expression levels or alter biochemical activity predispose humans and mouse models to a number of genetic diseases and cancer [5,6]. A 50 double flap containing a 30 unpaired nucleotide is the optimal substrate for Fen endonucleases from archaeal and eukaryotic organisms [8], whereas phage Fen1s are known to prefer pseudo-Y structures [7].
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