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Title: The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases

Abstract

OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC-Escherichia coli hosts, indicating that both the ATPase and nuclease activitiesmore » are required for OLD function in vivo.« less

Authors:
 [1]; ORCiD logo [1]; ORCiD logo [1]
  1. Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1604201
Resource Type:
Journal Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 48; Journal Issue: 5; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Schiltz, Carl J., Adams, Myfanwy C., and Chappie, Joshua S. The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases. United States: N. p., 2020. Web. doi:10.1093/nar/gkaa059.
Schiltz, Carl J., Adams, Myfanwy C., & Chappie, Joshua S. The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases. United States. doi:10.1093/nar/gkaa059.
Schiltz, Carl J., Adams, Myfanwy C., and Chappie, Joshua S. Mon . "The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases". United States. doi:10.1093/nar/gkaa059.
@article{osti_1604201,
title = {The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases},
author = {Schiltz, Carl J. and Adams, Myfanwy C. and Chappie, Joshua S.},
abstractNote = {OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC-Escherichia coli hosts, indicating that both the ATPase and nuclease activities are required for OLD function in vivo.},
doi = {10.1093/nar/gkaa059},
journal = {Nucleic Acids Research},
issn = {0305-1048},
number = 5,
volume = 48,
place = {United States},
year = {2020},
month = {2}
}