Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.
A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.
Institute for Biochemistry, Munich, Germany.
I F
IF
Moarefi
D
D
Small
I
I
Gilbert
M
M
Höpfner
S K
SK
Randall
C
C
Schneider
A A
AA
Russo
U
U
Ramsperger
A K
AK
Arthur
H
H
Stahl
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D016367:Baculoviridae; D001665:Binding Sites; D016213:Cyclins / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D009036:Moths; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism*; D013912:Threonine; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D016213:Cyclins; D004279:DNA, Viral; D013912:Threonine; D011494:Protein Kinases; D004265:DNA Helicases; D000409:Alanine