Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates.
Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5' flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5' ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.
SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, Fife, KY16 9SS, UK and Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife KY16 9SS, UK.
Timothy D
TD
Craggs
Richard D
RD
Hutton
Alfonso
A
Brenlla
Malcolm F
MF
White
J Carlos
JC
Penedo
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D061925:Immobilized Nucleic Acids / Q000032:analysis; D008958:Models, Molecular; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
D061925:Immobilized Nucleic Acids; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D045585:Flap Endonucleases