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Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen
N A Sunstrom1, N H Acheson, J A Hassell
1Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Abstract:
To map the DNA-binding domain of polyomavirus large T antigen, we constructed a set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in the large T antigen. Analysis of origin-specific DNA binding by mutant proteins expressed in Cos-1 cells revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu-398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggested that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116-amino-acid region between residues 282 and 398.
Insights
Researchers mapped the polyomavirus large T antigen DNA-binding domain. This crucial region, essential for viral DNA replication, spans amino acids 282 to 398.
Area of Science:
- Molecular Biology
- Virology
- Protein Biochemistry
Background:
- Polyomavirus large T antigen is essential for viral DNA replication.
- Understanding its DNA-binding domain is key to elucidating viral mechanisms.
Purpose of the Study:
- To precisely map the DNA-binding domain of the polyomavirus large T antigen.
- To identify the specific amino acid residues involved in origin DNA recognition.
Main Methods:
- Construction of plasmids encoding carboxy- and amino-terminal deletion mutants of large T antigen.
- Expression of mutant proteins in Cos-1 cells and analysis of origin-specific DNA binding.
- Evaluation of point mutants and internal deletions reported by other studies.
Main Results:
- The C-terminal boundary of the DNA-binding domain was identified at or near Glu-398.
- Fusion proteins lacking the first 200 N-terminal amino acids retained specific DNA binding.
- The N-terminal boundary was localized between amino acids 282 and 286, based on previous studies.
Conclusions:
- The DNA-binding domain of polyomavirus large T antigen is located within the 116-amino-acid region between residues 282 and 398.
- This defined region is critical for the specific recognition and binding of viral origin DNA.