Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase α

Abstract

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumorsuppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase α-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2×10⁸ M⁻¹. Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4×10⁸ M⁻¹, when RPA was coupled to the sensor chip via its smallest subunit, and 1×10⁸ M⁻¹, when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase α-primase complex (pol-prim) with a K_A value of 1×10¹⁰ M⁻¹. Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2×10¹⁰ M⁻¹ and 5×10⁹ M⁻¹, respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1 – 320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.