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Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen
Abstract:
Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH-terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2-terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen.
Insights
Simian virus 40 T antigen binds DNA more effectively when dephosphorylated or fragmented, particularly when the N-terminal region is removed. This suggests phosphorylation regulates T antigen
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Simian virus 40 tumor antigen (T antigen) plays a crucial role in viral DNA replication.
- Post-translational modifications, such as phosphorylation, can significantly alter protein function.
- Understanding T antigen's DNA-binding properties is key to deciphering viral replication mechanisms.
Purpose of the Study:
- To investigate the impact of phosphorylation and proteolysis on the DNA-binding affinity and kinetics of simian virus 40 T antigen.
- To identify the specific regions of T antigen responsible for DNA binding and how they are regulated.
Main Methods:
- DNA saturation experiments to measure T antigen binding to origin-containing DNA.
- Kinetic experiments to assess the rate of T antigen-DNA complex formation.
- Treatment of T antigen with alkaline phosphatase for dephosphorylation.
- Proteolytic digestion to generate T antigen fragments.
- Sedimentation analysis to evaluate protein oligomerization state.
Main Results:
- Dephosphorylated and proteolytically cleaved T antigen bound 1.5 to 2 times more origin-containing DNA than intact T antigen.
- These treatments also enhanced the rate of T antigen-DNA binding.
- Enhanced binding correlated with fragments lacking the N-terminal region.
- Dephosphorylation removed phosphates from both N-terminal and C-terminal regions without altering T antigen's monomeric state.
Conclusions:
- In vivo phosphorylation of the T antigen N-terminal region (residues 106-124) reduces its binding to the simian virus 40 DNA origin.
- This inhibitory effect is reversed by in vitro dephosphorylation or proteolysis.
- Phosphorylation may inactivate one of two distinct DNA-binding activities within T antigen.