Exonuclease subunit 2 (gp46) is the 560-amino-acid Rad50-family ABC-type ATPase subunit of the bacteriophage T4 Mre11-Rad50 (gp47-gp46) DNA end-processing complex. Containing split N- and C-terminal P-loop NTPase domains separated by a coiled-coil region with a central CXXC zinc-hook motif, gp46 assembles into a heterotetramer with the Mre11-like metallophosphoesterase nuclease subunit gp47. Binding to gp47 and double-stranded DNA ends stimulates gp46 ATP hydrolysis approximately 20-fold, driving conformational transitions that enable processive dsDNA exonucleolytic resection by gp47. Through this activity, gp46 is essential for homologous recombination, double-strand break repair, recombination-dependent DNA replication, and exonucleolytic degradation of fragmented host chromosomal DNA into mononucleotides for viral DNA synthesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000043 | MODIFY | Summary: gp46 is a Rad50-family P-loop NTPase that specifically binds and hydrolyzes ATP. Reason: Nucleotide binding (GO:0000166) is overly general; the specific ligand bound by the ABC-type nucleotide-binding domain of gp46 is ATP, captured by ATP binding (GO:0005524). Proposed replacements: ATP binding Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md Current model: gp46 **binds/hydrolyzes ATP at the Rad50 head dimer interface**; ATP-driven conformational cycling promotes assembly at DNA ends and transitions to a **translocation state** that supports processive nuclease action by gp47. |
| GO:0003677 DNA binding | IEA GO_REF:0000043 | ACCEPT | Summary: gp46 binds double-stranded DNA ends within the gp46/gp47 Mre11-Rad50 complex, and DNA binding strongly stimulates its ATPase cycle. Reason: Biochemical and structural analyses demonstrate that the Rad50-like head domain of gp46 directly contacts DNA substrates to couple ATP hydrolysis with nuclease translocation. Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md Gp46 is an ABC/SMC-like ATPase whose ATP turnover is **weak alone** but **strongly activated** in the presence of gp47 and dsDNA. |
| GO:0004518 nuclease activity | IEA GO_REF:0000043 | MODIFY | Summary: gp46 functions as part of the gp46/gp47 exonuclease complex that resects double-stranded DNA ends. Reason: Nuclease activity (GO:0004518) is broader than exonuclease activity (GO:0004527), which more specifically describes the DNA end-resection activity of the gp46/gp47 complex. Proposed replacements: exonuclease activity Supporting Evidence: file:BPT4/46/46-uniprot.txt FUNCTION: Exonuclease that plays a role in viral genome replication, DNA recombination, and host DNA degradation. file:BPT4/46/46-deep-research-falcon.md The **primary catalytic activity of the complex is 5′→3′ dsDNA exonuclease activity**, genetically assigned to gp46/gp47 and biochemically observed in reconstituted assays; **gp46 itself is also an ATP-binding/hydrolyzing enzyme** whose ATPase is strongly stimulated by gp47 and DNA. |
| GO:0004527 exonuclease activity | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: gp46 is an essential subunit of the T4 gp46/gp47 dsDNA exonuclease complex, though the metallophosphoesterase nuclease active site resides in gp47 while gp46 provides the ATPase motor activity required for processive resection. Reason: While the heterotetrameric gp46/gp47 complex catalyzes processive dsDNA exonucleolysis and requires gp46 ATP hydrolysis for processivity, gp46 itself is the Rad50-family ABC ATPase subunit rather than the catalytic nuclease subunit (gp47). Supporting Evidence: file:BPT4/46/46-uniprot.txt FUNCTION: Exonuclease that plays a role in viral genome replication, DNA recombination, and host DNA degradation. file:BPT4/46/46-deep-research-falcon.md Thus, gp46 is best annotated as the **ATPase/structural motor subunit** of an exonuclease complex rather than the nuclease active site itself. |
| GO:0005524 ATP binding | IEA GO_REF:0000043 | ACCEPT | Summary: gp46 binds ATP at its conserved Rad50-like P-loop nucleotide-binding domain dimer interface. Reason: ATP binding by gp46 is experimentally established and essential for both its ATPase activity and activation of the gp46/gp47 exonuclease complex. Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md Current model: gp46 **binds/hydrolyzes ATP at the Rad50 head dimer interface**; ATP-driven conformational cycling promotes assembly at DNA ends and transitions to a **translocation state** that supports processive nuclease action by gp47. |
| GO:0006281 DNA repair | IEA GO_REF:0000043 | MODIFY | Summary: gp46 is required for DNA double-strand break repair via homologous recombination during T4 infection. Reason: DNA repair (GO:0006281) is a general parent term; double-strand break repair (GO:0006302) is already annotated and more specifically captures the role of the gp46/gp47 MR complex in resecting double-strand breaks. Proposed replacements: double-strand break repair Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md gp46 is **essential in vivo** for **homologous recombination**, **double-strand break repair**, and **recombination-dependent DNA replication (RDR)** in bacteriophage T4. |
| GO:0006302 double-strand break repair | IEA GO_REF:0000002 | ACCEPT | Summary: As the Rad50 homolog of the T4 Mre11-Rad50 complex, gp46 is essential for resecting DNA double-strand breaks to initiate recombinational repair and recombination-dependent replication. Reason: Genetic and biochemical studies establish that gp46/gp47 is essential for processing double-strand breaks in bacteriophage T4. Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md gp46 is **essential in vivo** for **homologous recombination**, **double-strand break repair**, and **recombination-dependent DNA replication (RDR)** in bacteriophage T4. |
| GO:0006974 DNA damage response | IEA GO_REF:0000043 | MODIFY | Summary: gp46 participates in the response to DNA damage specifically by mediating double-strand break repair and recombinational fork restart. Reason: DNA damage response (GO:0006974) is a broad umbrella term; double-strand break repair (GO:0006302) is a more specific descendant term that accurately reflects the biological role of gp46. Proposed replacements: double-strand break repair Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md gp46 is **essential in vivo** for **homologous recombination**, **double-strand break repair**, and **recombination-dependent DNA replication (RDR)** in bacteriophage T4. |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MODIFY | Summary: gp46 is an enzyme that hydrolyzes ATP to drive conformational changes in the gp46/gp47 complex. Reason: Hydrolase activity (GO:0016787) is too broad; gp46 specifically catalyzes ATP hydrolysis (GO:0016887). Proposed replacements: ATP hydrolysis activity Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md Current model: gp46 **binds/hydrolyzes ATP at the Rad50 head dimer interface**; ATP-driven conformational cycling promotes assembly at DNA ends and transitions to a **translocation state** that supports processive nuclease action by gp47. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: gp46 is a Rad50-family ABC ATPase whose ATP hydrolysis activity is stimulated approximately 20-fold by gp47 and dsDNA and is required for processive DNA end resection. Reason: ATP hydrolysis activity (GO:0016887) is the primary intrinsic catalytic activity of gp46. Supporting Evidence: file:BPT4/46/46-deep-research-falcon.md Quantitatively, gp46 ATPase has **kcat ≈ 0.15 s⁻¹** alone and increases to **kcat ≈ 3.2 s⁻¹** with gp47 and dsDNA (≈20-fold activation), consistent with an ATP-driven conformational cycle in the assembled MR–DNA complex. |
| GO:0039657 symbiont-mediated suppression of host gene expression | IEA GO_REF:0000043 | MODIFY | Summary: gp46 is a subunit of the gp46/gp47 recombination nuclease complex that degrades cytosine-containing host chromosomal DNA; refine broad host gene expression suppression to degradation of host chromosome by virus. Reason: While immediate transcriptional shutoff of E. coli is initiated by Alc, AsiA, Alt, and ModA, the gp46/gp47 Mre11/Rad50-like nuclease complex degrades cytosine-containing host chromosomal DNA following Endonuclease II/IV cleavage, permanently eliminating the host template. The annotation is therefore best refined from GO:0039657 (symbiont-mediated suppression of host gene expression) to the specific mechanism GO:0099015 (degradation of host chromosome by virus). Proposed replacements: degradation of host chromosome by virus Supporting Evidence: file:BPT4/46/46-uniprot.txt FUNCTION: Exonuclease that plays a role in viral genome replication, DNA recombination, and host DNA degradation. |
| GO:0099015 degradation of host chromosome by virus | IEA GO_REF:0000043 | ACCEPT | Summary: Together with gp47, gp46 is required in vivo for the exonucleolytic degradation of cytosine-containing Escherichia coli DNA fragments into mononucleotides for viral DNA synthesis. Reason: Classic genetic and biochemical studies (Kutter & Wiberg 1968) established that mutants in genes 46 and 47 fail to convert endonuclease-cleaved host chromosomal DNA into acid-soluble nucleotides. Supporting Evidence: file:BPT4/46/46-uniprot.txt FUNCTION: Exonuclease that plays a role in viral genome replication, DNA recombination, and host DNA degradation. |
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Download this section (compressed HTML)Q: How do the recombination mediator UvsY and single-stranded DNA-binding protein gp32 remodel the conformational cycle of the gp46 ATPase head domain to switch gp47 from Mn2+-dependent 3'-5' exonucleolysis to Mg2+-dependent endonucleolytic resection?
Q: What is the structural basis for zinc-hook dimerization of the gp46 coiled-coil domains in bridging broken phage DNA ends vs tethering sister duplexes during recombination-dependent replication?
Experiment: Determine cryo-EM structures of the T4 gp46/gp47 heterotetramer bound to forked dsDNA substrates in the presence and absence of ATP-gamma-S, gp32, and UvsY.
Hypothesis: Binding of gp32 and UvsY stabilizes an open, endonuclease-competent conformation of the gp46/gp47 head assembly that exposes the 5'-terminated strand for endonucleolytic clipping near double-strand break ends.
Experiment: Perform single-molecule optical tweezers combined with confocal fluorescence tracking of fluorescently labeled gp46/gp47 complexes translocating on bacteriophage DNA ends at physiological Mg2+ and ATP concentrations.
Hypothesis: ATP hydrolysis by gp46 drives step-wise unidirectional translocation (~4 bp per ATP hydrolyzed) that is paused and redirected upon recruitment of UvsY and UvsX.
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