A SMALL PEPTIDE INHIBITOR OF DNA-REPLICATION DEFINES THE SITE OF INTERACTION BETWEEN THE CYCLIN-DEPENDENT KINASE INHIBITOR P21(WAF1) AND PROLIFERATING CELL NUCLEAR ANTIGEN

Background: p21(WAF1) is potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21(WAF1) can be induced by the human tumour suppressor protein p53, suggesting that the action of p21(WAF1) may be important in cancer prevention. We have investigated the interaction between p21(WAF1) and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21(WAF1) protein sequence. Results: We have established that the carboxy-terminal region of p21(WAF1) interacts with PCNA in a yeast two-hybrid screen. Interaction with p21(WAF1) involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21(WAF1) protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21(WAF1)). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. Conclusions: We have shown that PCNA and the cell-cycle regulator p21(WAF1) interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21(WFA1). Peptides of p21(WAF1) that interact with PCNA can inhibit DNA replication; such peptides or mimetics may thus prove useful in the treatment of hyperproliferative diseases, including cancer.