Human and Plant Proliferating‐Cell Nuclear Antigen have a Highly Conserved Binding Site for the p53–Inducible Gene Product p21^WAF1

Abstract

The mechanism(s) whereby higher plants respond to environmental agents that damage their DNA, which leads to the arrest of cell division, is poorly understood. In mammalian cells, the tumour-suppressor protein p53 plays a central role in a DNA-damage-induced cell-cycle-checkpoint pathway by induction of transcription of a set of gene products that have a direct role in a DNA-damage-induced cell-cycle growth arrest. One such protein, p21WAF', has been shown to be essential for radiation-induced growth arrest. There appear to be at least two cellular targets of p2IWAF1 during checkpoint control, the G,-cyclindependent kinases (CDK) and proliferating-cell nuclear antigen (PCNA). The aim of the research reported here was to determine whether the interactions between the human growth inhibitor p2IWAF1 and PCNA from plants and humans are conserved. If so, this would suggest that modulation of PCNA activity may play an important role in plant responses to DNA damage and would imply that functional homologue(s) of p21 exist in plants. We show that the p21 WAF1-interaction domain of PCNA is conserved between humans and plants. A peptide that contains the site of human p21WA11 that binds human PCNA has been used to precipitate PCNA from crude pea (Pisum savitum) extracts. We used the p2lWAF' peptide as an affinity matrix and showed that pea PCNA bound in a specific high-affinity manner. This finding was used to develop a purification protocol that allowed PCNA from plant tissue to be purified to homogeneity. Pure pea PCNA forms a stable complex with full-length human p2IWAF' and the specific amino acids of p2IwAF' required for the interaction have been identified. The critical residues were identical to those required for binding to human PCNA, which indicates that the interaction of human p21WAF' with PCNA is highly conserved at each amino acid position between pea and human.

Reversible Phosphorylation at the C-terminal Regulatory Domain of p21Waf1/Cip1 Modulates Proliferating Cell Nuclear Antigen Binding

“…They were then diluted 1:5 in lysis buffer containing 2.5 g/ml PCNA, and immunoprecipitation was carried out with AB1 as above. The immunoprecipitated complexes were separated by SDS-PAGE (42), transferred to nitrocellulose, and detected using AB1 (p21) and the polyclonal serum 3009 (PCNA (37)) following the method described in Ball and Lane (37).…”

“…We and others (37,39) have shown previously that a 20-amino acid peptide based on residues 140 -160 of p21 is sufficient to bind and form a high affinity interaction with PCNA that can be detected by peptide precipitation and ELISA. A quantitative ELISA was developed to compare PCNA binding to p21-derived phosphopeptides with PCNA binding to the unphosphorylated p21-based peptide.…”

“…PCNA binding was detected using the anti-PCNA sera 3009 (37). The data represent the mean of three experiments.…”

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

“…Hydrophobic residues of p21 shown in bold type occupy a hydrophobic pocket on the surface of PCNA when the two are complexed, as determined by structural analysis of the co-crystal (28). These residues of p21 have also been shown to be particularly important for interaction with PCNA by mutational analysis (27,29,30). Several residues in the PCNA-binding region of human p21 are identical or conservatively substituted in human FEN-1 and XPG, including the key hydrophobic residues Met 147 , Phe 150 , and Tyr 151 .…”

Differential post-translational modification of the tumour suppressor proteins Rb and p53 modulate the rates of radiation-induced apoptosis in vivo

“…50 ± 250 mg of tissue lysate was separated on 8, 10 or 15% SDS polyacrylamide gels as detailed in the ®gure legends. Western blot analysis was carried out as previously described (Ball and Lane, 1996).…”