Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region

Interaction between human Rap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X5X6F7(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.