A Complex between p53 Protein and DNA-Polymerase Alpha-Primase Displayed 3`-5` Exonuclease Activity
Recently it was found that an exonuclease activity is associated with the tumor suppressor protein p53 (Mummenbrauer et al., 1996, Cell 85, 1089-1099; Janus et al., 1999, Mol. Cell. Biol. 19, 2155-2168). The biochemical characteristics of this exonuclease pointed to a function as a proofreader (Huang, 1998, Oncogene 17, 261-270). Using surface plasmon resonance studies we could demonstrate that p53 forms a complex with the cellular replicative enzyme DNA polymerase alpha-primase complex (Kühn et al., 1999, Oncogene 18, 769-774). Here, we report on the purification and characterization of this five-subunits complex from baculovirus-infected insect cells. This complex possessed both DNA polymerase and 3'-5' exonuclease activities, which strongly supports the concept that p53 might fullfil an error-correcting function for this replicative DNA polymerase. The p53-containing five-subunits complex, but not the p53- and exonuclease-free form of the cellular polymerase was able to perform DNA synthesis starting from a 3'-mispaired primer end. The mismatched nucleotide was removed by the p53-associated exonuclease and exchanged by a correctly paired one by the DNA polymerase activity. Despite this, with a PhiX174am16-based reversion system, we were not able to observe a significant increase of the accuracy of DNA-polymerase alpha-primase in vitro. Thus, an important factor might be missing to couple p53's exonuclease to the DNA polymerase alpha-primase function.
C. Melle, A. Mohrdieck, H. Förster, H.-P. Nasheuer and F. Grosse
Institute for Molecular Biotechnology, Beutenbergstr. 11, D-07743 Jena, Germany