Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

Abstract

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.DOI: http://dx.doi.org/10.7554/eLife.21884.001

Structure of the processive human Pol δ holoenzyme

“…5d). 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] .…”

“…A 6× His-tag was introduced at the C-terminus of RPA1 subunit by PCR. The catalytically inactive FEN1-D181A was constructed as described previously 54 . Briefly, the gene sequence encoding WT human FEN1 was cloned via Gibson assembly into a pE-SUMO-pro expression vector (Lifesensors).…”

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…At the systems level, we see the importance of maintaining the B-DNA double helix structure, as potential non-B-DNA structures (PONDS) occur at high frequency in cancer as translocations and deletion break points (230). Furthermore, at RFs where the double helix is opened for processing, precise control of DNA ends is needed to avoid not only mutations but also template switching and large repeat expansions (231, 232). These and other data provide compelling support for the notion that DNA structures at forks, ends, and breaks are intrinsic risk factors that complexes such as MRN are under strong evolutionary pressure to tightly control.…”

Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities

“…We observed that DNA repair and replication enzymes typically sculpt their DNA substrates, as we found for nucleotide flipping by DNA repair glycosylases (Guan et al, 1998; Slupphaug et al, 1996; Thayer, Ahern, Xing, Cunningham, & Tainer, 1995), AP endonucleases (Tsutakawa et al, 2013), and endonuclease V (Dalhus et al, 2009), as well as for the flap exo- and endonuclease FEN1 (Rashid et al, 2017; Tsutakawa et al, 2017). Thus, part of our strategy to obtain specificity is to retain DNA binding but to block protein-mediated DNA conformational changes needed to position the substrate complex for catalysis.…”