QM/MM MD simulations on the enzymatic pathway of the human flap endonuclease (hFEN1) elucidating common cleavage pathways to RNase H enzymes

Flap endonucleases (FENs) are nucleic acid hydrolyzing enzymes in charge of excising 5?-small DNA and RNA fragments (flaps) protruding from nucleic acid structures during the lagging strand DNA replication or the long-patch base excision repair (LP-BER) processes. In this work we report, for the first time, an atomistic and energetic rendering of the enzymatic catalysis promoted by the human FEN1. After reconstruction of a reactive hFEN/double strand (ds) DNA adduct we employed mixed quantum-classical (QM/MM) metadynamics and umbrella sampling free energy calculations, with the QM part treated with the AM1/d-PhoT Hamiltonian, to perform an extensive characterization of all possible reaction pathways underlying the enzymatic cycle. Our extensive investigation points to a most likely reaction pathway very similar to that recently proposed for ribonuclease H, in which the rate-determining step is the nucleophilic attack of a water to the scissile phosphate, which occurs concomitantly with its activation by the pro-Rp oxygen of the nucleobase flanking the scissile phosphate. This step requires a free energy barrier in good agreement with experimental data (?G^?exp = 16.1 kcal/mol vs ? F^?calc = 16 ± 2 kcal/mol). Due to the important role of FENs in maintaining nucleic acid fidelity and cell proliferation, a detailed understanding of its enzymatic mechanism has broad interest to elucidate a key enzymatic biological process for preserving genome integrity and has implications for medical and biotechnological applications.