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 phas es of the cell cycle, and also binds to proliferating cell nuclear ant igen (PCNA), blocking DNA replication in vitro. Transcription of p21(W AF1) can be induced by the human tumour suppressor protein p53, sugges ting that the action of p21(WAF1) may be important in cancer preventio n. We have investigated the interaction between p21(WAF1) and PCNA usi ng a genetic two-hybrid screen and with arrays of synthetic peptides d erived from the p21(WAF1) protein sequence. Results: We have establish ed that the carboxy-terminal region of p21(WAF1) interacts with PCNA i n a yeast two-hybrid screen. Interaction with p21(WAF1) involves the c entral loop of PCNA, which connects the two domains of the PCNA monome r. The interaction was finely mapped using peptides derived from the e ntire sequence of the p21(WAF1) protein, and the critical residues wer e found to be QTSMTDFY (amino acids 144-151 of p21(WAF1)). Remarkably, a 20-residue peptide containing this sequence inhibited replication o f simian virus 40 (SV40) DNA in vitro and could capture PCNA from whol e cell extracts, demonstrating that small molecules can retain the bio logical activity characteristic of the whole protein. Sequential alani ne-scan mutations of the peptide demonstrated that its ability to bloc k replication correlates with its affinity for binding PCNA. Conclusio ns: We have shown that PCNA and the cell-cycle regulator p21(WAF1) int eract in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21(W FA1). Peptides of p21(WAF1) that interact with PCNA can inhibit DNA re plication; such peptides or mimetics may thus prove useful in the trea tment of hyperproliferative diseases, including cancer.