title: Physical and functional interactions of the tumor suppressor protein p53 and DNA polymerase alpha-primase.
abstract: The wild-type form of p53 contains an intrinsic 3'-5'-exonuclease activity. As p53 forms a complex with DNA polymerase alpha-primase (pol-prim) in vivo this finding suggests that p53 might cooperate with pol-prim to stabilize the genetic information of living cells. To test this hypothesis, exonuclease-free DNA pol-prim was expressed alone or together with p53 for purification. Pol-prim formed a complex with p53, which was purified by ion exchange and immunoaffinity chromatography from baculovirus-infected insect cells. The p53-containing pol-prim fractions removed a 3'-unpaired nucleotide with a 1.5-2-fold higher rate than a paired nucleotide, whereas the four subunit pol-prim did not have any exonuclase activity. Therefore, only p53/pol-prim was able to elongate a primer-template that contained a 3'-unpaired primer end in vitro. To achieve this, the 3'-5'-exonuclease activity of p53 excised the unpaired nucleotide at the 3'-end of the primer and created a paired 3'-end, which pol-prim was able to elongate. The exonuclease activity of p53 as well as the elongation of a primer with a mispaired 3'-end was inhibited specifically by the anti-p53 monoclonal antibodies PAb240 and PAb421.
authors.affiliation: Institut für Molekulare Biotechnologie e.V., Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Melle
authors.forename: Heinz-Peter
authors.initials: HP
authors.lastname: Nasheuer
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D011233:Precipitin Tests; D011485:Protein Binding; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D016159:Tumor Suppressor Protein p53; D004247:DNA; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I; D005092:Exonucleases