Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

Fahad Rashid, Paul D. Harris, Manal Zaher, Mohamed Abdelmaboud Sobhy, Luay Joudeh, Chunli Yan, Hubert Marek Piwonski, Susan E Tsutakawa, Ivaylo Ivanov, John A Tainer, Satoshi Habuchi, Samir Hamdan

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


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.
Original languageEnglish (US)
StatePublished - Feb 23 2017

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: The research reported here was supported by King Abdullah University of Science and Technology through core funding to S.M.H. and a Competitive Research Award (CRG3) to S.M.H. and J.A.T, as well as National Science Foundation CAREER award MCB-1149521 and National Institute of Health grant R01GM110387 to I.I. J.A.T. also acknowledges support of a Robert A. Welch Chemistry Chair, the Cancer Prevention and Research Institute of Texas, and the University of Texas System Science and Technology Acquisition and Retention STARs program. Computational resources were provided in part by a National Science Foundation XSEDE allocation CHE110042 and through an allocation at NERSC supported by the U.S. Department of Energy Office of Science contract DE-AC02-05CH11231. The authors declare no competing financial interests.


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