Endonuclease II (DenA; EndoII; EC 3.1.21.8) is a 136-amino-acid, magnesium-dependent GIY-YIG superfamily DNA endonuclease of bacteriophage T4 that initiates the degradation of unmodified, cytosine-containing host chromosomal DNA during early infection. Functioning as a dimer-of-dimers homotetramer that bridges two double-stranded DNA duplexes, DenA recognizes cytosine-rich sequence motifs (consensus 5'-GRCCG-3' / CCGC) and introduces single-strand nicks and double-strand breaks into the Escherichia coli chromosome while sparing T4 genomic DNA, which contains glucosylated 5-hydroxymethylcytosine. The resulting host DNA fragments are further processed into mononucleotides to supply precursors for phage DNA replication.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003677 DNA binding | IEA GO_REF:0000043 | ACCEPT | Summary: DenA directly binds cytosine-containing double-stranded DNA recognition sequences as a homotetramer bridging two DNA molecules. Reason: Electrophoretic mobility shift, footprinting, and crystallographic studies establish that DenA binds specific cytosine-containing dsDNA sequences to catalyze strand nicking. Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md Lagerbäck et al. show that EndoII behaves unusually among many GIY–YIG enzymes by forming an active **tetrameric** DNA complex that can bind **two DNA molecules** at once file:BPT4/denA/denA-uniprot.txt Sequence-specific endonuclease. Catalyzes nicking of the bottom strand of double-stranded DNA between the first and second base pair to the right of a top-strand CCGC motif. |
| GO:0004518 nuclease activity | IEA GO_REF:0000043 | MODIFY | Summary: Correct parent term that should be refined to the more specific GO:0004519 (endonuclease activity) already annotated to the gene. Reason: DenA is specifically an endonuclease (Endonuclease II, EC 3.1.21.8), which is captured by the descendant term GO:0004519 already present on this gene. Proposed replacements: endonuclease activity Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md **DenA (Endonuclease II; EndoII)** is a phage-encoded, sequence-context-sensitive DNA endonuclease of the **GIY–YIG nuclease superfamily** that initiates degradation (“restriction”) of **cytosine-containing, unmodified host DNA** during T4 infection. file:BPT4/denA/denA-uniprot.txt Sequence-specific endonuclease. Catalyzes nicking of the bottom strand of double-stranded DNA between the first and second base pair to the right of a top-strand CCGC motif. |
| GO:0004519 endonuclease activity | IEA GO_REF:0000120 | ACCEPT | Summary: Accurately captures the core catalytic activity of DenA (Endonuclease II, EC 3.1.21.8). Reason: DenA is a GIY-YIG superfamily DNA endonuclease that nicks and cleaves unmodified cytosine-containing double-stranded DNA. Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md **DenA (Endonuclease II; EndoII)** is a phage-encoded, sequence-context-sensitive DNA endonuclease of the **GIY–YIG nuclease superfamily** that initiates degradation (“restriction”) of **cytosine-containing, unmodified host DNA** during T4 infection. file:BPT4/denA/denA-uniprot.txt Sequence-specific endonuclease. Catalyzes nicking of the bottom strand of double-stranded DNA between the first and second base pair to the right of a top-strand CCGC motif. |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MODIFY | Summary: Overly broad parent term; DenA specifically functions as an endonuclease (GO:0004519). Reason: While phosphodiester bond cleavage is a hydrolase reaction, the more specific descendant term GO:0004519 (endonuclease activity) is already annotated to the gene. Proposed replacements: endonuclease activity Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md **DenA (Endonuclease II; EndoII)** is a phage-encoded, sequence-context-sensitive DNA endonuclease of the **GIY–YIG nuclease superfamily** that initiates degradation (“restriction”) of **cytosine-containing, unmodified host DNA** during T4 infection. file:BPT4/denA/denA-uniprot.txt Sequence-specific endonuclease. Catalyzes nicking of the bottom strand of double-stranded DNA between the first and second base pair to the right of a top-strand CCGC motif. |
| GO:0039657 symbiont-mediated suppression of host gene expression | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: Downstream consequence of host chromosome degradation rather than a direct transcriptional shutoff mechanism. Reason: DenA initiates nucleolytic breakdown of the host chromosome (GO:0099015), which secondarily eliminates templates for host gene expression, but DenA acts directly on DNA integrity rather than the transcription apparatus. Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md DenA/EndoII is positioned at the **initiation step** of T4 host DNA degradation, producing rare nicks in host DNA that enable further processing. file:BPT4/denA/denA-uniprot.txt Contributes to the degradation of host DNA, permitting the scavenging of host-derived nucleotides for phage DNA synthesis (PubMed:18539732, PubMed:19666720). |
| GO:0046872 metal ion binding | IEA GO_REF:0000120 | MODIFY | Summary: General term that should be refined to GO:0000287 (magnesium ion binding). Reason: DenA requires a catalytic divalent magnesium ion (Mg2+) bound at the conserved GIY-YIG active site (including Glu118), supporting refinement to GO:0000287 (magnesium ion binding). Proposed replacements: magnesium ion binding Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md A catalytic-surface residue **E118** is implicated in catalysis and also affects DNA binding: the **E118A** variant can bind stably as monomer/dimer to a single DNA substrate file:BPT4/denA/denA-uniprot.txt Name=Mg(2+); Xref=ChEBI:CHEBI:18420; |
| GO:0099015 degradation of host chromosome by virus | IEA GO_REF:0000043 | ACCEPT | Summary: Accurately captures the primary biological role of DenA during T4 infection. Reason: DenA catalyzes the initial endonucleolytic cleavage of unmodified cytosine-containing host chromosomal DNA in T4-infected Escherichia coli, initiating host chromosome breakdown for nucleotide scavenging. Supporting Evidence: file:BPT4/denA/denA-deep-research-falcon.md **DenA (Endonuclease II; EndoII)** is a phage-encoded, sequence-context-sensitive DNA endonuclease of the **GIY–YIG nuclease superfamily** that initiates degradation (“restriction”) of **cytosine-containing, unmodified host DNA** during T4 infection. file:BPT4/denA/denA-uniprot.txt Contributes to the degradation of host DNA, permitting the scavenging of host-derived nucleotides for phage DNA synthesis (PubMed:18539732, PubMed:19666720). |
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Download this section (compressed HTML)Q: What is the high-resolution structure of the DenA homotetramer bound simultaneously to two cytosine-containing dsDNA duplexes, and how does 5-hydroxymethylcytosine sterically or electronically prevent productive recognition?
Q: How does Ndd-mediated nucleoid peripheralization affect the accessibility of host chromosomal sites to DenA cleavage in vivo?
Experiment: Determine the cryo-EM or X-ray structure of the DenA(E118A) homotetramer in complex with two 5'-GRCCG-3' dsDNA substrates to resolve the DNA-bridging interface and base-recognition contacts.
Experiment: Perform genome-wide strand-break sequencing (END-seq or GLOE-seq) at early time points after T4 infection to map in vivo DenA cleavage hotspots across the Escherichia coli chromosome.
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