Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1
Fahad Rashid,
Paul D Harris,
Manal S Zaher,
Mohamed A Sobhy,
Luay I Joudeh,
Chunli Yan,
Hubert Piwonski,
Susan E Tsutakawa,
Ivaylo Ivanov,
John A Tainer,
Satoshi Habuchi,
Samir M Hamdan
Affiliations
Fahad Rashid
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Paul D Harris
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Manal S Zaher
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Mohamed A Sobhy
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Luay I Joudeh
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Chunli Yan
Department of Chemistry, Georgia State University, Atlanta, United States; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, United States
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Susan E Tsutakawa
Lawrence Berkeley National Laboratory, Berkeley, United States
Ivaylo Ivanov
Department of Chemistry, Georgia State University, Atlanta, United States; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, United States
John A Tainer
Lawrence Berkeley National Laboratory, Berkeley, United States; Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, United States
Satoshi Habuchi
Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.
