Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro.
The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.
Reynisdóttir
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Department of Biological Sciences, Columbia University, New York, New York 10027.
O'Reilly
D R
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Miller
L K
LK
Prives
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C
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics; D003850:Deoxyribonuclease I; D006367:HeLa Cells / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007313:Insecta; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology; D013696:Temperature
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I