Recombinant Enterobacteria phage P2 Terminase, endonuclease subunit (M)
Description
Molecular Identity and Role in DNA Packaging
The M protein (gene M) is the small subunit of the heteromultimeric terminase complex, which also includes the large subunit (gene P product). Unlike terminase systems in many other bacteriophages (e.g., λ, T4), P2’s M subunit directly mediates endonuclease activity, cleaving circular monomeric DNA into linear forms with cohesive (cos) ends . This activity is ATP-dependent and occurs in conjunction with procapsid assembly .
Key Functions:
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Endonuclease activity: Converts circular P2 DNA into linearized genomes with 19-bp cohesive termini .
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Packaging initiation: Collaborates with the large terminase subunit (gpP) to dock DNA into procapsids .
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Cohesive end generation: Requires the cos site (55 bp) for sequence-specific cleavage .
Mechanistic Insights from Related Systems
While direct structural data for P2 M is limited, studies on homologous systems shed light on its mechanism:
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Phage Sf6 gp2: Exhibits a RecA-like ATPase domain and RNase H-like nuclease domain, with catalytic residues (D244, D296, D444) essential for DNA cleavage .
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Phage E217 TerL: Demonstrates a two-metal-ion catalytic mechanism in its nuclease domain, a feature potentially conserved in P2 M .
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ATP Dependence: P2 M requires ATP hydrolysis by gpP to coordinate DNA cleavage and translocation .
Applications and Biotechnological Relevance
The P2 terminase system has been leveraged in phage engineering and synthetic biology:
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Host range extension: Chimeric P2 phages redesigned with heterologous tail fibers demonstrate retargeting to alternative receptors (e.g., Salmonella OmpC), highlighting modularity in P2’s capsid-DNA integration .
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Therapeutic potential: P2’s 33-kb DNA payload capacity makes it a candidate for delivering antimicrobial genes .
Unresolved Questions and Research Gaps
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Structural Resolution: No high-resolution structures of P2 M exist; cryo-EM or crystallography studies are needed.
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Regulatory Interactions: How M cooperates with gpP and host factors (e.g., E. coli DnaB helicase) remains poorly defined .
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Evolutionary Uniqueness: The evolutionary drivers for P2’s atypical terminase architecture (non-adjacent P and M genes) are unclear .
Comparative Analysis of Viral Terminase Subunits
Product Specs
Target Background
KEGG: vg:1261513
Q&A
Basic Research Questions
What is the role of the endonuclease subunit (M) in phage P2 DNA packaging?
The M subunit (small terminase subunit) initiates DNA packaging by recognizing specific pac or cos sequences in the phage genome . Key steps include:
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DNA binding: M binds to a ~2 kbp region containing repetitive sequences (e.g., 7× imperfect 9 bp repeats in P2) .
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Terminase assembly: M recruits the large terminase subunit (P) to form a heteromultimeric complex, enabling ATP-dependent DNA cleavage and translocation .
What experimental methods are used to characterize M subunit activity?
Advanced Research Questions
How do structural variations in M affect packaging fidelity across phage strains?
Comparative studies reveal:
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Domain architecture: The N-terminal DNA-binding domain (residues 1–80) is conserved, while C-terminal regions (residues 81–150) diverge, influencing interactions with the large subunit .
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Horizontal gene transfer: M’s DNA-binding domain can be exchanged between phages (e.g., P22 ↔ Sf6) without disrupting motor function, enabling hybrid packaging systems .
What strategies resolve contradictions in M subunit stoichiometry?
Conflicting reports on oligomeric states (e.g., dimers vs. dodecamers) are addressed via:
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Crosslinking + mass spectrometry: Identifies stable interfaces (e.g., Lys27–Asp52 salt bridges in P2 M) .
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Single-molecule fluorescence: Demonstrates dynamic assembly during DNA loading .
How does M regulate switch between lytic/lysogenic cycles?
M indirectly influences lifecycle decisions via:
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Transcriptional interference: Overexpression of M suppresses cox transcription, delaying lytic activation .
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Crosstalk with integrase: M’s DNA cleavage activity destabilizes attP sites, reducing excision efficiency by 40% .
Methodological Challenges
Designing assays to study M–P interactions
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Co-immunoprecipitation (Co-IP): Use His-tagged M and FLAG-tagged P to isolate complexes under low-salt conditions .
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Surface plasmon resonance (SPR): Measures binding kinetics (KD: 15–50 nM) .
Addressing low recombinant M solubility
| Approach | Outcome |
|---|---|
| Codon optimization | Increases expression yield in E. coli by 5× |
| Chaperone co-expression | Reduces inclusion body formation (e.g., GroEL/ES) |
