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

Oncogene. 1999 Jan 21;18(3):769-74. doi: 10.1038/sj.onc.1202327.

Abstract

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumor suppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase alpha-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 x 10(8) M(-1). Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4 x 10(8) M(-1), when RPA was coupled to the sensor chip via its smallest subunit, and 1 x 10(8) M(-1), when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase alpha-primase complex (pol-prim) with a K(A) value of 1 x 10(10) m(-1). Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2 x 10(10) m1(-1) and 5 X 10(9) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Polyomavirus Transforming / metabolism
  • DNA Polymerase I / metabolism*
  • DNA Primase / metabolism
  • DNA-Binding Proteins / metabolism
  • Humans
  • Mice
  • Replication Protein A
  • Surface Plasmon Resonance
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Antigens, Polyomavirus Transforming
  • DNA-Binding Proteins
  • RPA1 protein, human
  • Replication Protein A
  • Rpa1 protein, mouse
  • Tumor Suppressor Protein p53
  • DNA Primase
  • DNA Polymerase I