Flap endonuclease-1 (FEN-1) play important roles in DNA replication and repair. In this study, the kinetics parameters of mutants at highly conserved aromatic residues, Tyr³³, Phe⁷⁹, and Phe²⁷⁸Phe²⁷⁹, in the vicinity of the catalytic centers of the FEN-1's molecules were examined. According to the kinetic parameters of the mutants and other results, DNA binding model of the phFEN-1 with the nick substrate was proposed as shown in the figure. The stacking interactions of Tyr³³ and Phe⁷⁹ might play important roles to fix the template strand and the downstream strand, in close proximately to the active center to form the productive transient state leading to the hydrolysis.