MODULATION OF THE DNA BINDING ACTIVITY OF SV40 LARGE T ANTIGEN BY CELLULAR PROTEIN PHOSPHATASE 2Ac (REPLICATION PROTEIN C)

Abstract

Simian virus 40 (SV40) large tumor antigen (T Ag) binds to two contiguous sites at the SV40 origin of replication, referred to as sites I and II. Of these two sites, only site II is required for replication. T Ag binds with highest efficiency to site I but site II . is the functional binding site for the initiation of SV40 DNA replication in vivo and in vitro. At 37°C, the temperature essential for the initiation of SV40 DNA replication in vitro, ATP is required for the interaction of T Ag and Site ll but not for its interaction with Site I. A replication protein, Replication Protein C (RP-C), has been shown to dephosphorylate specific phosphoamino acid residues on the T Ag molecule (Virshup and Kelly 1989). RP-C was purified from HeLa cells by its ability to stimulate reconstituted replication in vitro. This protein was shown to be the catalytic subunit of protein phosphatase 2A. In this study, the interaction between T Ag and Site ll DNA has been studied in the presence and absence of RP-C. The protein-DNA interactions have been analyzed by three methods - nitrocellulose filter binding assays, DNA mobility shift assays, and potassium permanganate footprinting assays. The binding affinity of T Ag for Site II can be stimulated two fold by the addition of RP-C. Qualitative changes in the T Ag-Site ll interaction were also detected in the presence of RP-C.