Direct interaction of Gadd45 with PCNA and evidence for competitive interaction of Gadd45 and p21Waf1/Cip1 with PCNA.
We have previously shown (Smith et al., 1994) that antibodies raised against the growth arrest and DNA damage inducible protein Gadd45 co-precipitate proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. Here we demonstrate that Gadd45 can directly bind to PCNA using a Far-western blotting approach. In this assay, a Gadd45 bacterial expression vector was modified to allow synthesis of purified 32P-labeled Gadd45 fusion protein. This protein was used to detect filter bound PCNA protein, while filter bound Gadd45 protein could also be detected by free PCNA molecules. Using recombinant proteins in conjunction with immunoprecipitation and immunoblotting, we show that Gadd45 competes with p21 for binding to PCNA and conversely, p21 blocks the ability of Gadd45 to bind PCNA. In addition, p21 appears to disrupt PCNA trimers whereas Gadd45 has a lesser effect. PCNA trimer disruption was also observed in UV-irradiated cells but not in repair-defective xeroderma pigmentosum group A (XP-A) cells.
D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western / Q000379:methods; D003110:Colonic Neoplasms / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism* / Q000494:pharmacology; D004249:DNA Damage; D004347:Drug Interactions; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008175:Lung Neoplasms / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000187:drug effects / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D014466:Ultraviolet Rays; D000097642:GADD45 Proteins
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D011994:Recombinant Proteins; D000097642:GADD45 Proteins; C493956:CDKN1A protein, human