Pausing in simian virus 40 DNA replication by a sequence containing (dG-dA)27.(dT-dC)27

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• A specific DNA sequence, (dG-dA)27.(dT-dC)27, was identified as a sequence limiting DNA amplification.
• This sequence was found within a region affecting polyoma virus integration in rat cells.

Purpose of the Study:

• To investigate the functional impact of inserting a (dG-dA)n.(dT-dC)n repeat sequence into the simian virus 40 (SV40) genome.
• To determine the effect of this insertion on viral replication and DNA synthesis.

Main Methods:

• Cloning of a 200 bp sequence containing a 54 bp (dG-dA)27.(dT-dC)27 repeat into the SV40 genome.
• Transfection of the modified SV40 DNA into African Green monkey kidney CV1 cells.
• Isolation of variant viruses via plaque purification and characterization through growth rate, titer, plaque size, and mixed infection experiments.
• Pulse labeling experiments to analyze DNA replication fork progression and nucleotide incorporation rates.

Main Results:

• The inserted sequence was stably maintained in the SV40 genome.
• Variant viruses exhibited slower growth, lower titers, and smaller plaques compared to wild-type SV40.
• Mixed infections showed the variant virus to be stable, but wild-type replicated faster.
• Pulse labeling revealed the inserted sequence acts as a pause site for DNA replication forks, with a pause duration of approximately one minute.

Conclusions:

• The synthetic alternating purine-pyrimidine sequence functions as a replication pause site within the SV40 genome.
• This replication pause significantly impacts viral kinetics, leading to reduced growth and lower viral titers.
• The findings provide insights into the role of specific DNA sequences in regulating DNA replication dynamics.