Human and plant proliferating-cell nuclear antigen have a highly conserved binding site for the p53-inducible gene product p21WAF1

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, p21WAF1, has been shown to be essential for radiation-induced growth arrest. There appear to be at least two cellular targets of p21WAF1 during checkpoint control, the G1-cyclin-dependent 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 p21WAF1 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 p21WAF1 exist in plants. We show that the p21WAF1-interaction domain of PCNA is conserved between humans and plants. A peptide that contains the site of human p21WAF1 that binds human PCNA has been used to precipitate PCNA from crude pea (Pisum savitum) extracts. We used the p21WAF1 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 p21WAF1 and the specific amino acids of p21WAF1 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 p21WAF1 with PCNA is highly conserved at each amino acid position between pea and human.