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Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin
Abstract:
We isolated revertants of a polyomavirus whose origin of DNA replication contains a point mutation in the palindrome to which large T antigen binds. Four independent second-site revertants contain an Asp-286----Asn-286 substitution in large T antigen. This mutant large T antigen activates replication of DNAs containing the mutant polyomavirus origin as well as replication of DNAs containing the wild-type origin; however, replication of DNAs with enhancer mutations is not activated by this large T antigen. The Asn-286 mutation occurs in a positively charge region of large T antigen near the location of several mutations which inactivate DNA replication. We suggest that this region of large T antigen is responsible for recognition of specific DNA sequences at the origin and that ionic forces are important for this interaction.
Insights
We identified a mutation in polyomavirus large T antigen that restores DNA replication. This mutant T antigen recognizes both mutated and wild-type origins, suggesting ionic forces are key for DNA sequence recognition.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Polyomavirus DNA replication is initiated at a specific origin.
- Large T antigen is essential for viral DNA replication.
- Mutations in the origin or T antigen can disrupt replication.
Purpose of the Study:
- To investigate the role of large T antigen in polyomavirus DNA replication.
- To identify mutations in large T antigen that can restore replication from a mutated origin.
- To understand the mechanism of DNA origin recognition by large T antigen.
Main Methods:
- Isolation and characterization of polyomavirus revertants.
- Site-directed mutagenesis of the polyomavirus origin and large T antigen.
- Replication assays using wild-type and mutant viral DNA.
Main Results:
- Four independent revertants showed an Asp-286 to Asn-286 substitution in large T antigen.
- The Asn-286 mutant T antigen replicated DNA from both mutant and wild-type origins.
- Replication of DNAs with enhancer mutations was not activated by the mutant T antigen.
- The mutation occurred in a positively charged region of T antigen, near known replication-inactivating mutations.
Conclusions:
- The Asn-286 mutation in large T antigen restores its ability to initiate DNA replication.
- This region of large T antigen is critical for recognizing specific DNA sequences at the replication origin.
- Ionic interactions likely play a significant role in the binding of large T antigen to the DNA origin.