| Evidence type | Key finding | Quantitative details/statistics | Source (author year journal) and URL |
|---|---|---|---|
| In vivo cleavage mapping | T4 **denA** encodes Endonuclease II (EndoII), which cleaves cytosine-containing plasmid DNA at sequence-dependent sites in infected cells; cleavage occurs around a central variable base pair and yields blunt ends or short 5' overhangs. Consensus motifs derived from mapped sites were **5'-GRCCGCNTYGC-3'** and weighted **5'-CGRCCGCNTTGSYNGC-3'**. (pqac-00000011, pqac-00000025, pqac-00000034) | Cleavage at **12 sites** in pBR322; starts **before 10 min postinfection** at 37°C; site-specific cleavage frequencies ranged **10–90% by 50 min**; sequenced sites showed **25–65%** cleavage; products had **0–2 bp** strand displacement / **1–2 nt 5' overhangs**. (pqac-00000011, pqac-00000009, pqac-00000010) | Carlson et al. 1993, *J. Biol. Chem.* (1993-04). https://doi.org/10.1016/S0021-9258(18)52959-8 |
| In vitro binding/oligomerization | EndoII is a **GIY-YIG nuclease** that binds DNA predominantly as a **tetramer engaging two DNA substrates**; free enzyme is mainly dimeric at low ionic strength and shifts toward tetramers at higher salt. The catalytic-surface mutant **E118A** uniquely forms monomer/dimer complexes on a single DNA. (pqac-00000021, pqac-00000026, pqac-00000028, pqac-00000032) | Expected masses: monomer **16.8 kDa**, dimer **33.6 kDa**, tetramer **67.2 kDa**; gel filtration showed ~**35 kDa** (dimer), ~**74.7 kDa** (tetramer), and ~**170 kDa** for tetramer + two DNAs (expected ~**179 kDa**); stable binding required **>30 bp** DNA; ~**80%** of K12A C4-complexed 44-bp substrate was nicked after **15 min** in-gel with MgCl2; example enzyme:DNA ratios included **42:1** and **27:1–450:1**. (pqac-00000027, pqac-00000028, pqac-00000026) | Lagerbäck et al. 2009, *Nucleic Acids Res.* (2009-08). https://doi.org/10.1093/nar/gkp652 |
| Infection timing/role | DenA/EndoII performs the **initial nicking step in host DNA degradation** during T4 takeover, targeting host cytosine DNA while T4 DNA is protected by hydroxymethylcytosine/glucosylation. Nicked regions then become substrates for **DenB (Endonuclease IV)** and downstream **46/47 exonuclease** processing, supporting irreversible host shutoff and nucleotide scavenging for phage DNA synthesis. (pqac-00000024, pqac-00000014, pqac-00000017) | Host DNA degradation is mainly observed between about **6–20 min** postinfection; nucleotide-production/DNA-replication enzymes are produced around **3–8 min**; in stationary-phase infections with a DNA polymerase amber mutant, **40%** of host 3HdT became acid-soluble by **60 min** and **60%** by **120 min**; with wild-type T4, only about **10%** reduction was seen by **60 min** because released nucleotides were rapidly reincorporated into phage DNA. (pqac-00000017, pqac-00000016) | Kutter et al. 2018, *Viruses* (2018-07). https://doi.org/10.3390/v10070387 |
| Context effects | EndoII recognizes a **16-bp variable site**, but cleavage efficiency depends strongly on both local sequence/shape and longer-range DNA context; recognition appears to involve both base readout and sequence-dependent DNA structure. Introducing a strong synthetic site suppresses cleavage at nearby weaker natural sites, implying competition over an extended DNA region. (pqac-00000008, pqac-00000033) | Efficient synthetic motif: **CGRCCGCNTTGGCNGC**; a 16-bp variable sequence was sufficient for cleavage, but a “best-base-at-each-position” consensus was paradoxically cleaved poorly; insertion of a synthetic site reduced cleavage at natural sites within about **800–1,500 bp**; Lagerbäck et al. proposed interference over roughly **~1000 bp** on each side for neighboring sites in mode II cleavage models. (pqac-00000008, pqac-00000033) | Carlson et al. 1996, *J. Bacteriol.* (1996-11). https://doi.org/10.1128/jb.178.22.6419-6426.1996; Lagerbäck et al. 2009, *Nucleic Acids Res.* (2009-08). https://doi.org/10.1093/nar/gkp652 |


*Table: This table summarizes the main experimental evidence supporting functional annotation of Enterobacteria phage T4 denA/Endonuclease II, including cleavage specificity, oligomeric state, infection role, and context dependence. It is useful as a compact evidence map linking key findings to quantitative observations and primary sources.*