T4 DNA
Description
Introduction to T4 DNA
T4 DNA refers to the double-stranded DNA genome of bacteriophage T4, a virulent Escherichia coli-infecting virus. As a model system in molecular biology, T4 has provided foundational insights into DNA replication, repair, and packaging. Its genome spans approximately 168,903 base pairs (bp) and encodes 289 genes, including enzymes like T4 DNA ligase and polymerase, which are critical for viral replication and widely used in biotechnology .
Key Genomic Features
The T4 genome is modular, containing clusters of genes for DNA metabolism (e.g., DNA ligase, polymerase), structural proteins, and replication initiation factors. Its terminal redundancy enables circular permutation during packaging .
Origins of Replication
T4 DNA replication initiates bidirectionally from two primary origins (oriA and oriB):
Replication involves host-derived RNA polymerase for priming and T4-encoded helicases (gp41) and sliding clamps (gp45) for processivity .
DNA Packaging Dynamics
T4’s packaging motor (terminase complex) exhibits remarkable force generation and speed :
| Parameter | Value | Significance |
|---|---|---|
| Packaging Force | >60 pN | Overcomes DNA electrostatic repulsion |
| Translocation Rate | Up to 2,000 bp/s | Completes 171-kb genome packaging in <10 minutes |
| ATP Turnover | >300 s⁻¹ | High-energy coordination with replication |
Dynamic velocity changes and reversible pausing allow synchronization with transcription and repair .
Applications in Molecular Biology
T4 DNA ligase and polymerase are indispensable tools:
T4 ligase’s activity is inhibited by excess dsDNA (>100 ng/μl), likely due to competitive binding .
Recent Advances and Engineering
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Chimeric Ligases: Fusion of T4 ligase’s DBD with thermostable archaeal ligases improves blunt-end ligation efficiency .
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Motor Protein Engineering: Modifications to T4’s packaging motor aim to enhance synthetic DNA delivery systems .
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Polymerase Optimization: T4 DNA polymerase variants reduce large deletions (≥3 bp) in CRISPR edits by 60% .
Product Specs
Product Science Overview
T4 DNA Ligase is responsible for catalyzing the formation of phosphodiester bonds between adjacent nucleotides in DNA. This process is essential for repairing breaks or “nicks” in the DNA backbone that can occur during DNA replication, recombination, and repair. The enzyme works by joining the 3’-hydroxyl (OH) end of one nucleotide with the 5’-phosphate (P) end of another, effectively sealing the gap and restoring the integrity of the DNA molecule .
The mechanism of T4 DNA Ligase involves three main steps :
- Enzyme Adenylation: The enzyme first binds to a molecule of adenosine triphosphate (ATP) and transfers an adenosine monophosphate (AMP) group to itself.
- AMP Transfer: The AMP group is then transferred to the 5’-phosphate end of the DNA at the site of the nick.
- Phosphodiester Bond Formation: The 3’-hydroxyl group of the adjacent nucleotide attacks the 5’-phosphate-AMP complex, resulting in the formation of a phosphodiester bond and the release of AMP.
T4 DNA Ligase is widely used in various molecular biology applications, including :
- Cloning: The enzyme is used to ligate DNA fragments into vectors, such as plasmids, to create recombinant DNA molecules. This is a critical step in the cloning process, allowing for the insertion of genes or other DNA sequences into vectors for propagation and expression in host cells.
- DNA Repair: T4 DNA Ligase is employed in DNA repair assays to study the mechanisms of DNA damage and repair.
- Library Construction: The enzyme is used in the construction of genomic and cDNA libraries, where it helps to ligate DNA fragments into vectors for sequencing and analysis.
- Site-Directed Mutagenesis: T4 DNA Ligase is used to introduce specific mutations into DNA sequences by ligating oligonucleotides containing the desired mutations into target DNA.
The recombinant form of T4 DNA Ligase offers several advantages over the native enzyme :
- High Purity: Recombinant production allows for the purification of the enzyme to a high degree of purity, reducing the risk of contamination with other proteins or nucleases.
- Consistency: Recombinant production ensures a consistent and reliable supply of the enzyme, with uniform activity and performance across different batches.
- Cost-Effectiveness: Large-scale production of recombinant T4 DNA Ligase is more cost-effective than isolating the enzyme from natural sources, making it more accessible for research and industrial applications.
