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:

Rashid, Fahad ^[1]; Harris, Paul D. ^[1]; Zaher, Manal S. ^[1]; Sobhy, Mohamed A. ^[1]; Joudeh, Luay I. ^[1]; Yan, Chunli ^[2]; Piwonski, Hubert  ^[1];

• Search DOE PAGES for author "Piwonski, Hubert"
• Search DOE PAGES for ORCID "0000-0001-8666-3945"

Tsutakawa, Susan E. ^[3]; Ivanov, Ivaylo ^[2]; Tainer, John A. ^[4]; Habuchi, Satoshi ^[1]; Hamdan, Samir M.  ^[1]

• Search DOE PAGES for author "Hamdan, Samir M."
• Search DOE PAGES for ORCID "0000-0001-5192-1852"

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+ Show Author Affiliations

1. Division of Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
2. Department of Chemistry, Georgia State University, Atlanta, United States, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, United States
3. Lawrence Berkeley National Laboratory, Berkeley, United States
4. Lawrence Berkeley National Laboratory, Berkeley, United States, Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, United States

Publication Date:

Thu Feb 23 00:00:00 EST 2017

Research Org.:

Lawrence Berkeley National Lab (LBNL) Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC); King Abdullah University of Science and Technology; National Science Foundation (NSF); National Institutes of Health (NIH) Clinical Center

OSTI Identifier:

1347470

Alternate Identifier(s):

OSTI ID: 1347471; OSTI ID: 1628865

Grant/Contract Number:  

AC02-05CH11231; 2201 CRG3; MCB-1149521; R01GM110387

Resource Type:

Published Article

Journal Name:

eLife

Additional Journal Information:

Journal Name: eLife Journal Volume: 6; Journal ID: ISSN 2050-084X

Publisher:

eLife Sciences Publications, Ltd.

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Life Sciences & Biomedicine - Other Topics