p53 in complex with DNA is resistant to ubiquitin-dependent proteolysis in the presence of HPV-16 E6
Molinari, M.; Milner, J.
Oncogene 10(9): 1849-1854
1995
ISSN/ISBN: 0950-9232 PMID: 7753560 Document Number: 453710
The tumour suppressor p53 is a transcription factor with high affinity for specific DNA target sequences. Wild type p53 has a very short half life in normal cells but the protein shows transient accumulation in response to DNA damage, accompanied by up-regulation of target genes such as p21 and induction of growth arrest in G1 of the cell cycle. The rapid turnover of p53 may involve the ubiquitin-dependent proteolytic pathway. In order to investigate p53 turnover we have employed an in vitro system with rabbit reticulocyte lysate, in which ubiquitin dependent degradation of p53 is mediated by the oncoprotein E6 of human papilloma virus type 16 (HPV-16). Using this system we have previously shown that E6-mediated degradation is preferential for p53 in the 1620+ conformation (reactive with the monoclonal antibody PAb1620). p53-1620+ is a pre-requisite for specific DNA binding and we have now asked if p53 in complex with DNA remains susceptible to ubiquitin dependent proteolysis in the presence of E6. Our results indicate that p53-DNA complexes are resistant to degradation, whereas the 'free' protein is completely degraded within 20 min. Moreover, E6 did not complex with p53-DNA, possibly due to masking of sites recognised either by E6 or by the E6-associated protein (E6-AP) which facilitates E6-p53 interaction. Preincubation with E6 inhibited the DNA binding capacity of p53 and this effect could be explained, at least in part, by ubiquitination of the p53 protein.