title: Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1.
abstract: 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.
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Fahad
authors.initials: F
authors.lastname: Rashid
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Harris
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Manal S
authors.initials: MS
authors.lastname: Zaher
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Sobhy
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Luay I
authors.initials: LI
authors.lastname: Joudeh
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, United States.|Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, United States.
authors.forename: Chunli
authors.initials: C
authors.lastname: Yan
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Hubert
authors.identifier: 0000-0001-8666-3945
authors.initials: H
authors.lastname: Piwonski
authors.affiliation: Lawrence Berkeley National Laboratory, Berkeley, United States.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Tsutakawa
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, United States.|Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, United States.
authors.forename: Ivaylo
authors.initials: I
authors.lastname: Ivanov
authors.affiliation: Lawrence Berkeley National Laboratory, Berkeley, United States.|Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, United States.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Habuchi
authors.affiliation: Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
authors.forename: Samir M
authors.identifier: 0000-0001-5192-1852
authors.initials: SM
authors.lastname: Hamdan
mesh_terms: D004247:DNA / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D000072760:Single Molecule Imaging; D013379:Substrate Specificity
chemical_list: D004247:DNA; D045585:Flap Endonucleases; C468317:FEN1 protein, human
keywords: Human purified proteins; biochemistry; biophysics; expressed proteins in E. coli; none; recombinant protein; structural biology