Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates

摘要

Flap endonuclease 1 (Fen1) is a highly conservedudstructure-specific nuclease that catalyses audspecific incision to remove 50 flaps in doublestrandedudDNA substrates. Fen1 plays an essentialudrole in key cellular processes, such as DNA replicationudand repair, and mutations that compromiseudFen1 expression levels or activity have severeudhealth implications in humans. The nucleaseudactivity of Fen1 and other FEN family members canudbe stimulated by processivity clamps such asudproliferating cell nuclear antigen (PCNA); however,udthe exact mechanism of PCNA activation is currentlyudunknown. Here, we have used a combination ofudensemble and single-molecule Fo¨rster resonanceudenergy transfer together with protein-induced fluorescenceudenhancement to uncouple and investigateudthe substrate recognition and catalytic steps of Fen1udand Fen1/PCNA complexes. We propose a model inudwhich upon Fen1 binding, a highly dynamic substrateudis bent and locked into an open flap conformationudwhere specific Fen1/DNA interactions can be established.udPCNA enhances Fen1 recognition of the DNAudsubstrate by further promoting the open flap conformationudin a step that may involve facilitatedudthreading of the 50 ssDNA flap. Merging our dataudwith existing crystallographic and molecularuddynamics simulations we provide a solution-basedudmodel for the Fen1/PCNA/DNA ternary complex.