Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference
Abstract:
Essential nucleotide contacts between the SV40 large T (tumor) antigen and binding sites I and II on the SV40 genome have been inferred from in vitro methylation- and ethylation-interference experiments. Each site contains two clusters of guanine residues that reduce the specific binding of T antigen when modified. Methylation at any one of nine guanines within site I or any one of five guanines within site II severely interferes with the interaction of T antigen with each respective site. Methylation at any one of a second group of five guanines within site II results in an appreciably weaker effect on the binding of T antigen. A similar inhibitory effect on binding is observed upon ethylation of adjacent phosphate residues. Although there are significant differences in the nucleotide sequence of the two binding sites, the pattern of protein contacts is strikingly similar between sites I and II. Three-dimensional projection reveals that the guanine contacts within each binding site are localized so that the specific binding interactions are accessible from only one face of the DNA helix.
Insights
Simian virus 40 (SV40) large T antigen binding sites were mapped using chemical modifications. Guanine residues in both sites are crucial for T antigen interaction, with contacts accessible from one DNA helix face.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The simian virus 40 (SV40) large T (tumor) antigen plays a critical role in viral replication and oncogenesis.
- Understanding the precise interactions between viral proteins and their genomic binding sites is fundamental to virology and cancer research.
Purpose of the Study:
- To identify essential nucleotide contacts between SV40 large T antigen and its specific binding sites (sites I and II) on the SV40 genome.
- To elucidate the structural basis of T antigen recognition and binding to its cognate DNA sequences.
Main Methods:
- In vitro methylation and ethylation interference assays were employed to probe nucleotide-protein interactions.
- Chemical modifications of guanine residues and phosphate backbones were used to assess their impact on T antigen binding.
Main Results:
- Specific guanine residues within both binding sites I and II were identified as critical for SV40 large T antigen interaction.
- Methylation of nine guanines in site I or five guanines in site II severely impaired T antigen binding, indicating essential contacts.
- Ethylation of adjacent phosphate residues also interfered with T antigen binding, suggesting backbone involvement.
Conclusions:
- Despite sequence variations, SV40 binding sites I and II exhibit remarkably similar patterns of guanine contacts with large T antigen.
- These critical guanine contacts are spatially localized on one face of the DNA helix, facilitating specific protein-DNA recognition.
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