Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen.
In an attempt to distinguish simian virus 40 (SV40) large T antigen (T) binding to ATP from hydrolysis, specific mutations were made in the ATP-binding site of T according to our model for the site (M. K. Bradley, T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster, Proc. Natl. Acad. Sci. USA 84:4026-4030, 1987). Two acidic residues predicted to make contact with the magnesium phosphate were changed to alanines. The mutated T gene was completely defective for viral DNA synthesis and for virion production, and it was dominant defective for viral DNA replication. The defective T gene encoded a stable product (2905T) that oncogenically transformed mouse cell lines. 2905T, immunoprecipitated from transformed-cell extracts, bound SV40 origin DNA specifically and, surprisingly, it was active as an ATPase. A recombinant baculovirus was constructed for the production and purification of the mutant protein for detailed biochemical analyses. 2905T had only 10% of the ATPase and helicase of wild-type T. The Km of 2905T for ATP in ATPase assays was the same as the Km of wild-type T. ATP activated the ATPase activity of wild-type T, but not of 2905T. As tested by gel bandshift assay, 2905T bound to SV40 origin DNA and to individual sites I and II with affinities similar to that of the wild type. However, ATP did not modulate the DNA-binding activity of mutant T to site II. Therefore, this mutation in the ATP-binding site in T resulted in defects in the interaction between the protein and ATP that appeared to be responsible for the determination of the active state of T for DNA binding versus ATPase.
Weiner
B M
BM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Bradley
M K
MK
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000378:metabolism; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011487:Protein Conformation; D013539:Simian virus 40 / Q000235:genetics* / Q000254:growth & development; D013329:Structure-Activity Relationship; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases