cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen

Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.

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