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DNA binding of polyomavirus large T-antigen: kinetics of interactions with different types of binding sites
K Bondeson1, O Rönn, G Magnusson
1Department of Medical Immunology and Microbiology, Uppsala University, Biomedical Centre, Sweden.
Abstract:
Polyomavirus large T-antigen binds to GRGGC sites in double-stranded viral DNA, regulating transcription and replication. Using surface plasmon resonance to record interactions of large T-antigen with different types of binding sites, we found that the configuration of recognition motifs influenced both the association and dissociation rates. Particularly, the complex formed at the origin of DNA replication was labile. A comparison of the interactions between large T-antigen and binding sites with one, two and four GRGGC motifs in tandem showed a strong preference for dimer binding, without detectable co-operativity between dimers. Sodium chloride stabilised the complexes, whereas the dissociation increased rapidly by increasing pH above 7.0.
Insights
Polyomavirus large T-antigen binding to viral DNA is influenced by the arrangement of recognition sites. The DNA replication origin complex is unstable, and binding is favored by dimers without cooperativity.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Polyomavirus large T-antigen is crucial for viral transcription and replication.
- This protein binds to specific DNA sequences known as GRGGC motifs.
Purpose of the Study:
- To investigate how the configuration of GRGGC binding sites affects the kinetics of large T-antigen interaction.
- To characterize the binding properties at the viral DNA replication origin.
Main Methods:
- Surface plasmon resonance was employed to measure the association and dissociation rates of large T-antigen with various DNA binding sites.
- Binding affinities were assessed for sites with single, tandem dimeric, and tetrameric GRGGC motifs.
Main Results:
- The arrangement of GRGGC motifs significantly impacts the association and dissociation rates of large T-antigen.
- The complex formed at the DNA replication origin demonstrated lability.
- A preference for dimer binding was observed, with no significant cooperativity between dimers.
- Sodium chloride stabilized the complexes, while elevated pH (>7.0) increased dissociation.
Conclusions:
- The structural configuration of DNA recognition sites is a key determinant of polyomavirus large T-antigen binding dynamics.
- Understanding these interactions provides insights into viral gene regulation and replication mechanisms.