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Flap endonucleases (FENs) are structure-specific enzymes that play critical roles in DNA replication and repair. Three members of the FEN family have been investigated during this project in complexes with DNA substrates and metal ions: bacteriophage T5FEN, hFEN and Trypanosoma brucei FEN. T5FEN wild type and two catalytically inactive versions, D153K and D155K were successfully crystallized in complexes with DNA substrates containing 5' or 3' overhangs. The crystal structure for T5FEN-D153K in complex with a duplex containing 5' overhangs at each end and two Mg2+ ions was solved. The structure of T5FEN-D155K was solved in complex with a duplex containing 3' overhangs and a Ca2+ ion in the active site. In addition, wild type T5FEN was also crystallized with the same 3'-overhang substrate in the absence of metal ions. These structures revealed that the single strand-5' overhang in T5FEN-D153K pushed electrostatically and looped-up before threading through an arch-like structure composed of two helixes located over the active site of the enzyme. This arch is fully ordered in all of these structures. In the active sites of the variant complexes the Lys-153 and Lys155 are visible. The lysine's long side chain allows its ε amino group to occupy similar positions to the metal ion sites in one of the two active site subsites known as Cat1. The ε amino of Lys-153 directly coordinates the scissile phosphate of the DNA substrate. Important residues concerned with the 5' overhang threading are also determined. His-36 rotates by 180° to allow the duplex DNA movement before threading while Tyr-90 and Phe-105 form a gate-like structure after the 5' overhang has threaded. The conserved Arg-86 plays a critical role in 5' overhang transmission during threading process. Lys-83 and Arg-125 are found to interact symmetrically with the DNA backbone in the T5FEN-D155K:DNA complex. A new trans-arch/distal phosphate-binding site composed of Gly- 70 and Lys-71 has been determined in the far side of the arch. These structures of T5FEN also have a binding site for the potassium ion within the H3TH motif coordinated by the main chain carbonyl oxygens of three residues and directly interacted with the DNA phosphate group.