Base unpairing at the flap junction controls the rate of FEN1‐catalyzed cleavage of DNA during replication and repair

Flap Endonuclease 1 (FEN1) recognizes and cleaves 5′ single‐stranded DNA flaps to create nicked duplex products during DNA metabolic processes such as Okazaki fragment maturation, long‐patch base excision repair and telomere maintenance, and therefore has an essential role in genome duplication and stability (1). Defects in human FEN1 are associated with cancer and other genetic diseases (2). Hence, there is considerable interest in hFEN1 as a therapeutic target and diagnostic or prognostic biomarker (3–5), which can be aided by a detailed mechanistic understanding of the enzyme. Previous studies have shown that hFEN1 bends its DNA substrate, and suggest that unpairing of two bases in the duplex beyond the 5′ flap helps position the scissile phosphate in the active site for cleavage (6,7). These studies have largely been performed under equilibrium conditions and in the absence of Mg 2+ , which is indispensible for catalysis. To study the hFEN1 mechanism under catalytically competent conditions, we utilized transient kinetic approaches and monitored individual events in the millisecond time scale, including DNA binding and bending, base unpairing and cleavage, which elucidated the order of events and rate‐determining step(s) in the reaction. Furthermore, we examined the role of several conserved active‐site residues in the reaction mechanism. A FRET assay revealed that hFEN1 binds and bends the DNA substrate rapidly at a diffusion‐limited rate (10 9 M −1 s −1 ), indicating that these events occur simultaneously. Lack of Mg 2+ and mutations in the active site do not affect this binding/bending rate, thus this initial event does not depend on specific contacts within the catalytic pocket. A 2‐aminopurine fluorescence‐based assay revealed unpairing of base(s) at the flap junction at a much slower rate of 20 s −1 . Importantly, quench‐flow measurements revealed that base unpairing limits the cleavage rate to 20 s −1 . A subsequent event, likely related to product release, limits the overall steady state turnover rate to 1 s −1 . Mutations in active site residues D34, Y40, K93, R100, E160 and D181 exhibit differential effects on base unpairing and cleavage. The results show that base unpairing is a pre‐requisite for flap cleavage, and clarify the contributions of these conserved residues toward an electrostatic environment that stimulates precise cleavage of the 5′ flap. The findings also apply to other 5′‐nucleases that share similar active site residues and a two‐metal ion dependent reaction mechanism (8,9).