Studying the mechanism of human flap endonuclease 1 — A key enzyme in DNA replication and repair

Flap Endonuclease 1 (FEN1) recognizes and cleaves 5' single-stranded DNA flaps during DNA replication and repair to keep genome stability. Additionally, it can also process exonuclease (EXO) and gap-endonuclease substrates. Homozygous knockout of FEN1 is embryonically lethal in mice, and FEN1 defects and FEN1 over-expression have been associated with numerous types of cancers, autoimmune diseases, and chronic inflammation in humans. Thus, there is considerable interest in human FEN1 (hFEN1) as a therapeutic target and diagnostic biomarker, which can be assisted by a detailed mechanistic understanding of the enzyme. Previous studies have shown that both DNA substrate and hFEN1 undergoes universal and local conformational changes to reach a catalytically adept state. New crystal structures of DNA-bound hFEN1 provide novel ideas about roles that conformational changes of DNA and hFEN1 may play in the reaction mechanism. For instance, past biochemical studies of DNA conformation done under non-catalytic conditions with Ca2+ have implied that base unpairing is a crucial step in the reaction, but the new structural data indicate otherwise. To clarify the mechanism under catalytically competent circumstances, I utilized transient kinetic approaches and monitored individual events (from substrate binding to product release) on the millisecond timescale. I found that hFEN1 binds and bends the DNA substrate at a diffusion-limited rate, indicating that these events co-occur. Lack of Mg2+ and mutations in the active site do not affect this binding/bending rate. Thus this initial step does not depend on specific contacts within the catalytic pocket. A 2- aminopurine fluorescence-based assay and quench-flow measurements revealed that DNA distortion at the junction is a pre-requisite for flap cleavage and limits the cleavage rate to 25 s-1. A subsequent event, nicked product release, limits the steady-state turnover rate to 1.5 s-1. Past studies highlight several critical conserved active site residues: Asp-34 and Asp-181 coordinate metal ions; Lys-93 and Arg-100 navigate the 5'-flap through the active site and interact with the scissile phosphate; and Tyr-40 stacks against the +1 base to the scissile phosphate. Mutations in these residues disrupt DNA distortion and cleavage, suggesting that active site residues with different roles in the hFEN1 catalytic mechanism all contribute to the electrostatic environment and rate-limiting conformational changes that enable effective and precise cleavage of 5'-flaps. Beyond hFEN1, these findings may help us to understand other structure-specific nucleases that are important for resolving different types of intermediate DNA structures formed during replication, repair and recombination.