Title: Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

Abstract

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'–3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. Furthermore, the striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNAmore » binding, suggesting a structural basis for its processivity.« less

Authors:

Shi, Yuqian ^[1]; Hellinga, Homme W. ^[1]; Beese, Lorena S. ^[1]

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+ Show Author Affiliations

1. Duke Univ. Medical Center, Durham, NC (United States)

Publication Date:

Mon May 22 00:00:00 EDT 2017

Research Org.:

Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Org.:

National Institutes of Health (NIH)

OSTI Identifier:

1373796

Grant/Contract Number:  

R01 GM091487; P01 CA092584

Resource Type:

Accepted Manuscript

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Volume: 114; Journal Issue: 23; Journal ID: ISSN 0027-8424

Publisher:

National Academy of Sciences

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Rad2/XPG superfamily; DNA repair; flap endonuclease; crystallography; exonuclease