Structural basis of 5′ flap recognition and protein-protein interactions of human flap endonuclease 1

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5 ' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an alpha-helix forming the helical gateway of hFEN1 recognizes the 5 ' flap prior to its threading into the active site for cleavage. We also found that the beta-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the beta-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.