Electron microscopic analysis of partially replicated bacteriophage T7 DNA

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophage T7 DNA replication is initiated from specific origins.
• UV irradiation introduces damage that can impede DNA replication and affect genetic processes.
• Cross-reactivation is a phenomenon where genetic markers are transferred between related viruses.

Purpose of the Study:

• To analyze the replication patterns of partially replicated bacteriophage T7 DNA after UV irradiation.
• To investigate the distribution of replication forks and eye forms in T7 DNA molecules.
• To understand how UV damage influences T7 DNA replication and cross-reactivation.

Main Methods:

• Isolation and electron microscopic analysis of partially replicated T7 DNA from infected Escherichia coli.
• Alignment of replication structures (eye forms, forks) to the left end of the T7 genome.
• Annealing of 32P-labeled progeny DNA from UV-irradiated phage with ordered restriction fragments of T7 DNA.

Main Results:

• Replication was observed to occur only in the left 25-30% of the T7 DNA molecules.
• Analysis supported partial-replica hypotheses, indicating bidirectional replication from origins towards UV damage.
• Photoreactivation studies suggested thymine dimers are the primary UV damage blocking cross-reactivation at the right end of the genome.

Conclusions:

• Bacteriophage T7 DNA replication proceeds bidirectionally from origins, with efficiency influenced by UV damage.
• The extent of replication correlates with marker rescue efficiency during cross-reactivation.
• Thymine dimers are identified as significant UV-induced lesions hindering replication and cross-reactivation.