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

Abstract

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 -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 -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 -pin region in hFEN1 protein interactions that are essential for DNA replication and repair.