Abstract

Structure-specific endonucleases (SSEs) cleave the DNA substrate in a precise position based on the specific DNA 3D structure. Human flap endonuclease 1 (hFEN1) is a 5′ SSE that prevents DNA instability by processing Okazaki fragment 5′-flaps with remarkable efficiency and selectivity using two-metal-ion catalysis. Recent structural and mutagenesis data of hFEN1 suggest that phosphate steering favors specificity and catalysis. Here, we investigate the phosphate steering mechanism at the atomistic level using microsecond-long molecular dynamics and well-tempered metadynamics simulations of wild-type and mutant systems of hFEN1. We show how positively charged second and third-shell residues operate the phosphate steering mechanism to promote catalysis through (i) substrate recruitment; (ii) precise cleavage localization; and (iii) substrate release, thus actively preventing the off-target incision of the substrate. Importantly, structural comparisons of hFEN1 and other nuclease enzymes suggest that phosphate steering may also serve the structure-based selection of the specific DNA substrate by other 5′ structure-specific nucleases.

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