Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.
We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.
Ray
S
S
Department of Microbiology, State University of New York, Stony Brook 11794-8621.
Anderson
M E
ME
Loeber
G
G
McVey
D
D
Tegtmeyer
P
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D001483:Base Sequence; D002474:Cell-Free System; D004265:DNA Helicases / Q000378:metabolism; D004252:DNA Mutational Analysis; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D011487:Protein Conformation; D013539:Simian virus 40 / Q000235:genetics*
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D000251:Adenosine Triphosphatases; D004265:DNA Helicases