Structure-specific 5’-nucleases are highly conserved phosphodiesterases that recognize a diverse range of DNA and RNA structures and therefore play a central role in all aspects of DNA metabolism. It is of significant interest to understand how these highly conserved proteins recognize and cleave a diverse range of DNA structures. Human flap endonuclease 1 (hFEN1), the typified member of 5’-nucleases, recognizes an ideal substrate of a double stranded-DNA bearing a double flap nick-junction that consists of a 5’ flap DNA or RNA of various lengths and a one nucleotide 3’ DNA flap. The cell produces over 50 millions of these double flap structures during cell division and 10³-10⁴ per day during long patch base excision repair.

Recent substrate and product structures of hFEN1, in the absence of the 5’-flap, propose that binding at the base of the flap junction induces DNA bending providing a platform for verifying the 3’ and 5’ flap structures. Using single molecule fluorescence resonance energy transfer (FRET) we characterize the dynamics of DNA bending by hFEN1 and reveal a highly complex mechanism that interrogates the structures of the 3’ and 5’ flaps prior to committing to DNA bending. The engagement of the active site metal ions optimizes the structure of the bent substrate and facilitates the unpairing of the two-nucleotides flanking the scissile phosphate for cleavage. Our results consolidate the disputed biochemical and structural data and suggest that substrate specificity among 5’-nucleases might result from their specific requirement to induce the two common intermediary steps, DNA bending and two-nucleotides unpairing.