Immunopurified Rb protein inhibits SV40 T antigen-dependent stimulation of DNA polymerase alpha.

Abstract:

It has been shown that purified SV40 large T antigen (Tag) forms a complex with both human and calf thymus DNA polymerase alpha and stimulates its activity. Furthermore, Tag has also been found to complex with purified human Rb protein. Here, we show the effect of Rb protein on the stimulation of DNA polymerase alpha by Tag, in an in vitro system using either purified human or calf thymus DNA polymerase alpha and either primed single-stranded M13 DNA or calf thymus-activated DNA. Both human and calf thymus enzymes were dose-dependently stimulated several fold by Tag. The stimulation was also observed in the coupled reaction of primase and polymerase alpha, using unprimed single-stranded M13 DNA. These stimulatory effects were, however, completely abolished by preincubating Tag with an equimolar amount of Rb protein. Primase activity of DNA polymerase alpha-primase complex was also stimulated by Tag, and this stimulation was abolished by the presence of Rb protein. In contrast, free primase was not affected by either Tag or Rb protein. Kinetic analysis revealed that in the presence of Tag the apparent Km for the template of either human or calf DNA polymerase alpha was decreased by approximately 2.5-fold and the Vmax was increased twofold, whereas Tag complexed with Rb protein did not affect the Km or the Vmax. These results suggest a competition between Rb protein and DNA polymerase alpha for binding to Tag, which may be a key step for the initiation of SV40 DNA replication.

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