Protein Old (overcoming lysogenization defect) is a 583-amino-acid Class 1 OLD family antiphage defense enzyme of Escherichia phage P2 comprising an N-terminal ABC-type ATPase domain and a C-terminal Toprim two-metal-ion nuclease domain. Expressed from the P2 prophage, Old exhibits 5'-to-3' double-stranded DNA exonuclease and single-stranded DNA/RNA nuclease activities coupled to ATP hydrolysis, mediating abortive infection that blocks bacteriophage lambda replication and causes lethality in recBC-deficient Escherichia coli hosts.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000043 | MODIFY | Summary: Broad parent term of ATP binding, which is already annotated on this record. Reason: Broad keyword-derived term; Old specifically binds and hydrolyzes ATP via its N-terminal ABC ATPase domain, which is captured by GO:0005524 (ATP binding). Proposed replacements: ATP binding Supporting Evidence: PMID:32009148 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. |
| GO:0004518 nuclease activity | IEA GO_REF:0000043 | MODIFY | Summary: Broad parent nuclease term; Old is experimentally characterized as a double-stranded DNA 5'-3' exonuclease. Reason: Superseded by the specific experimentally supported descendant term GO:0051908 (double-stranded DNA 5'-3' DNA exonuclease activity) already present on this record. Proposed replacements: double-stranded DNA 5'-3' DNA exonuclease activity Supporting Evidence: PMID:7836278 The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. |
| GO:0004527 exonuclease activity | IEA GO_REF:0000043 | MODIFY | Summary: Broad parent exonuclease term; Old specifically exhibits double-stranded DNA 5'-3' DNA exonuclease activity. Reason: Superseded by the specific descendant term GO:0051908 (double-stranded DNA 5'-3' DNA exonuclease activity) already present on this record. Proposed replacements: double-stranded DNA 5'-3' DNA exonuclease activity Supporting Evidence: PMID:7836278 The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. |
| GO:0005524 ATP binding | IEA GO_REF:0000043 | ACCEPT | Summary: Old contains a canonical N-terminal ABC-family ATP-binding domain with conserved Walker A, Walker B, D-loop, and H-loop motifs required for in vivo function. Reason: Structural and mutational studies confirm that the N-terminal ABC ATPase domain of P2 Old binds and hydrolyzes ATP. Supporting Evidence: PMID:32009148 Guided by our structural findings and sequence conservation (Supplementary Figure S6), we identified the putative catalytic side chains in P2 OLD and engineered substitutions in the critical ABC ATPase motifs (K37A, Walker A; D299A, Walker B; H306A, D-loop; H332A, H-loop) and the Toprim metal binding and nuclease residues (E398A, D453A and D455A, metal A; E402, D541 and E543, metal B; K550A). |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MODIFY | Summary: Broad parent enzyme class; Old specifically catalyzes double-stranded DNA 5'-3' exonucleolytic cleavage and ATP hydrolysis. Reason: Broad keyword-derived term that should be refined to the two specific hydrolytic activities of P2 Old: GO:0051908 (double-stranded DNA 5'-3' DNA exonuclease activity) and GO:0016887 (ATP hydrolysis activity). Proposed replacements: double-stranded DNA 5'-3' DNA exonuclease activity ATP hydrolysis activity Supporting Evidence: PMID:7836278 The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. PMID:32009148 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. |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | ACCEPT | Summary: Old coordinates two catalytic divalent metal ions (sites A and B) in its C-terminal Toprim nuclease domain as well as Mg2+ in its ABC ATPase active site. Reason: Site-directed mutagenesis of metal-A (E398, D453, D455) and metal-B (E402, D541, E543) coordinating residues in the Toprim domain abolishes P2 Old function in vivo. Supporting Evidence: PMID:32009148 Guided by our structural findings and sequence conservation (Supplementary Figure S6), we identified the putative catalytic side chains in P2 OLD and engineered substitutions in the critical ABC ATPase motifs (K37A, Walker A; D299A, Walker B; H306A, D-loop; H332A, H-loop) and the Toprim metal binding and nuclease residues (E398A, D453A and D455A, metal A; E402, D541 and E543, metal B; K550A). |
| GO:0051908 double-stranded DNA 5'-3' DNA exonuclease activity | EXP PMID:7836278 The old exonuclease of bacteriophage P2. | ACCEPT | Summary: Purified P2 Old fusion protein processively degrades double-stranded DNA in the 5'-to-3' direction from 5'-phosphoryl or 5'-hydroxyl termini, releasing 5'-phosphoryl mononucleotides. Reason: Direct biochemical characterization in PMID:7836278 demonstrates processive 5'-to-3' dsDNA exonuclease activity. Supporting Evidence: PMID:7836278 The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides. |
| GO:0051908 double-stranded DNA 5'-3' DNA exonuclease activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC-mapped annotation reinforcing the experimentally demonstrated 5'-to-3' dsDNA exonuclease activity of P2 Old. Reason: Consistent with the experimental annotation from PMID:7836278. Supporting Evidence: PMID:7836278 The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides. |
| GO:0016887 ATP hydrolysis activity | IMP PMID:32009148 The full-length structure of Thermus scotoductus OLD defines... | NEW | Summary: P2 Old contains an N-terminal ABC-family ATPase domain whose ATP hydrolysis activity is strictly required for Old function in vivo. Reason: Substitutions in the conserved Walker A (K37A), Walker B (D299A), D-loop (H306A), and H-loop (H332A) ATPase catalytic motifs abolish P2 Old activity in vivo. Supporting Evidence: PMID:32009148 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. |
| GO:0006308 DNA catabolic process | IDA PMID:7836278 The old exonuclease of bacteriophage P2. | NEW | Summary: P2 Old processively degrades double-stranded and single-stranded DNA into 5'-phosphoryl mononucleotides. Reason: Biochemical assays show processive 5'-to-3' exonucleolytic degradation of DNA by purified P2 Old. Supporting Evidence: PMID:7836278 The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides. |
| GO:0051607 defense response to virus | IMP PMID:4913204 Bacteriophage lambda; abortive infection of bacteria lysogen... | NEW | Summary: Expressed from the P2 prophage, Old mediates abortive infection that blocks superinfecting bacteriophage lambda growth by more than six orders of magnitude. Reason: P2 Old is a canonical prophage-encoded antiphage defense system that aborts superinfection by bacteriophage lambda. Supporting Evidence: PMID:4913204 The efficiency of plating of wild-type lambda on a host lysogenic for P2 is less than 10(-6), and only a small number of infected cells produce progeny phage. PMID:2695400 The old (overcoming lysogenization defect) gene product of bacteriophage P2 kills Escherichia coli recB and recC mutants and interferes with phage lambda growth |
| GO:0030430 host cell cytoplasm | NAS PMID:32009148 The full-length structure of Thermus scotoductus OLD defines... | NEW | Summary: Old functions in the Escherichia coli host cell cytoplasm to sense and cleave nucleic acids during phage interference. Reason: P2 Old is a soluble cytoplasmic nuclease-ATPase expressed in the Escherichia coli host cytoplasm. Supporting Evidence: PMID:32009148 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. |
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Download this section (compressed HTML)Q: How does inhibition of host RecBCD by bacteriophage lambda Gam (or genetic loss of recBC) trigger the conformational switch in P2 Old from an autoinhibited ATP-bound state to an active DNA/tRNA-cleaving nuclease?
Experiment: Perform cryo-EM analysis of full-length P2 Old in the presence of branched DNA repair intermediates and ATP analogs to determine how RecBCD-substrate accumulation allosterically gates the Toprim active site.
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