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 alanin e 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 T12 4A T antigen in greater detail by using purified protein from a baculo virus expression system. Purified T124A is defective in SV40 DNA repli cation in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein f orms double-hexamer complexes at the origin in an ATP-dependent fashio n, although the binding reaction requires somewhat higher protein conc entrations than the wild-type protein. Binding of T124A protein result s in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication de fect of T124A protein is not due to failure to recognize and occupy th e origin. Under some conditions T124A is capable of unwinding short or igin DNA fragments. However, the mutant protein is almost completely d efective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially iden tical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibl y as a result of altered hexamer-hexamer interactions. The phenotype o f T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activi ty of T antigen in infected cells.