Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase alpha.
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.
Kühn
C
C
Institut für Molekulare Biotechnologie, Abt. Biochemie, Jena, Germany.
Müller
F
F
Melle
C
C
Nasheuer
H P
HP
Janus
F
F
Deppert
W
W
Grosse
F
F
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D051716:Replication Protein A; D020349:Surface Plasmon Resonance; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*