Functional sites of human PCNA which interact with p21 (Cip1/Waf1), DNA polymerase δ and replication factor C

Background: PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21 (Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary. Results: Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. analysis of their functions, along the previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase δ (polδ) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and polδ-interaction sites. Competition between p21 and polδ or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol δ DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. Conclusions: Semi-saturated amounts of p21 selectively block formation of the active pol δ complex but not the RFC-PCNA complex at 3'- ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.