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Salt-stable binding of the large T protein to DNA in polyoma virus chromatin
Abstract:
Nucleoprotein complexes extracted from the nuclei of mouse cells lytically infected with polyoma virus contain an ATPase activity which appears to correspond to that of the viral large T protein, as it exhibits the same characteristic properties; in particular, the activity is extensively inhibited by polyclonal antibodies from animals bearing polyoma tumors (anti-T antigen antibodies) and by monoclonal antibodies against large T. Significant amounts of DNA were immunoprecipitated by adding these antibodies to the nucleoprotein complex, suggesting that the protein is tightly bound to DNA in the viral chromatin. Since one of the monoclonal antibodies quantitatively immunoprecipitated the pulse-labeled replicative intermediates, we conclude that some large T protein remains physically associated with the DNA throughout its replication cycle. After exposure to salt concentrations higher than 1 M KCl, about half of the large T-specific ATPase activity was still observed to co-sediment with 21S form I viral DNA. The observations that the sedimentation coefficient of the salt-stable complexes was shifted to 16S after a limited endonucleolytic digestion, and that both the viral DNA and the ATPase activity were co-precipitated in the presence of polyethylene glycol at high ionic strength, further demonstrated that the protein is engaged in an unusually stable complex with DNA in the viral chromatin.
Insights
Polyoma virus large T protein possesses ATPase activity and remains bound to viral DNA throughout replication. This protein forms a stable complex with DNA within the viral chromatin, even under high salt conditions.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Polyoma virus infection involves viral proteins interacting with host cell machinery.
- The large T protein is a key viral factor in polyoma virus replication.
Purpose of the Study:
- To investigate the biochemical properties and DNA-binding characteristics of the polyoma virus large T protein's ATPase activity.
- To determine if the large T protein remains associated with viral DNA during its replication cycle.
Main Methods:
- Extraction of nucleoprotein complexes from infected mouse cells.
- Characterization of ATPase activity using specific antibodies (anti-T antigen and anti-large T monoclonal antibodies).
- Immunoprecipitation assays to detect DNA-protein interactions.
- Co-sedimentation and co-precipitation studies under varying salt concentrations and after enzymatic digestion.
Main Results:
- The viral large T protein exhibits characteristic ATPase activity, inhibited by specific antibodies.
- Significant amounts of viral DNA were immunoprecipitated with anti-T antigen and anti-large T antibodies, indicating tight DNA binding.
- Pulse-labeled replicative intermediates were immunoprecipitated, showing large T protein association throughout replication.
- The large T-specific ATPase activity remained associated with viral DNA even after exposure to high salt concentrations (>1 M KCl).
- Salt-stable complexes containing viral DNA and ATPase activity were characterized by sedimentation analysis and co-precipitation.
Conclusions:
- The polyoma virus large T protein possesses an intrinsic ATPase activity crucial for viral replication.
- Large T protein remains physically associated with viral DNA throughout its replication cycle.
- An unusually stable complex exists between the large T protein and viral DNA within the viral chromatin.