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.
Highlights
Overcoming lysogenization defect (OLD) proteins comprise a poorly characterized family of nucleases that contain an N-terminal ATPase domain and C-terminal Topisomerase/Primase (Toprim) catalytic domain [1,2].OLD homologs are widely conserved in bacteria, archaea and some viruses and can be subdivided into two classes [3]
We described the full-length structure of the Class 1 OLD homolog from Thermus scotoductus, which reveals a three domain architecture consisting of an N-terminal ATP-binding cassette (ABC) ATPase domain, a dimerization domain, and a C-terminal Toprim domain
The Ts OLD ATPase domain is topologically related to the nucleotide binding domain (NBD) of genome maintenance proteins (Supplementary Figure S9)
Summary
Overcoming lysogenization defect (OLD) proteins comprise a poorly characterized family of nucleases that contain an N-terminal ATPase domain and C-terminal Topisomerase/Primase (Toprim) catalytic domain [1,2]. OLD homologs are widely conserved in bacteria, archaea and some viruses and can be subdivided into two classes [3]. Class 1 OLD homologs exist as single, isolated genes while Class 2 old genes always appear in tandem with a UvrD/PcrA/Rep-like helicase. The Class 1 OLD homolog from the temperate bacteriophage P2 remains the best characterized to date. Genetic studies showed old+ P2 lysogens kill Escherichia coli recB and recC mutant hosts after infection and interfere with bacteriophage growth [4,5]. Further in vitro characterization of recombinant P2 OLD revealed DNA exonuclease activity and ribonuclease activity [6]. A saturating genomewide transposon screen of Salmonella typhimurium indicated that the old gene is conditionally essential in some instances like temperature stress [7], but the underlying mechanism for this phenotypic observation has yet to be clarified
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