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

Journal Article · 02 February 2020 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkaa059 · OSTI ID:1596875

Schiltz, Carl J. ^[1]; Adams, Myfanwy C.  ^[1]; Chappie, Joshua S.  ^[1]

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1. Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA

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 activities are required for OLD function in vivo.

• Sponsoring Organization: USDOE; National Institutes of Health (NIH)
• Grant/Contract Number: AC02-06CH11357
• OSTI ID: 1596875
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 5 Vol. 48; ISSN 0305-1048
• Publisher: Oxford University PressCopyright Statement
• Country of Publication: United Kingdom
• Language: English