A conserved loop–wedge motif moderates reaction site search and recognition by FEN1

Abstract

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3′-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3′-flap. This recognition event allosterically signals hydrolytic removal of the 5′-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved ‘wedge’ residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated ‘loop–wedge’ mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3′-flap verification to function.

Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations

“…FEN1, involved in resolution of 5′ flaps in lagging-strand replication, is one of the most well-studied in the family and can serve as an example to broadly understand 5′ nuclease mechanisms. In FEN1, incision requires at least a four-step process of: 1) Initial binding to dsDNA regions of the 5′ flap; 2) specific substrate validation of target features (dsDNA, single-strand 5′ flap, and a 1-nt 3′ flap) with concomitant conformation shifts in both the protein and DNA conformations; 3) shifting the scissile phosphate into the catalytic site; and finally 4) two-metal-based phosphodiester bond cleavage (29,(35)(36)(37)(38)(39). Thus, only after substrate testing by coordinated DNA and protein conformational changes (35) is FEN1 licensed to cut DNA, making it distinct from other nuclease classes where substrate validation is at the binding step.…”

A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1

“…A key feature of the duplex binding site of the FEN/EXO family enzymes is the H2TH motif that binds a monovalent ion, generally K + . It has recently been proposed that FEN1 makes its initial contact with its DNA substrate via the H2TH domain ( 42 ). We have now found that Ct GEN1 has a closely similar structure, yet its conformation of depends upon the nature of the bound cation.…”

Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level

“…Consequently, FEN1 can actively create a 3′ flap in the non-cognate SF-6,0 substrate. This mechanism would explain the 1 nt shift of the cleavage site in SF versus DF substrates [55] , [95] .…”