Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cle aves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antig en (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking , binding, or cleavage is enhanced by PCNA, we tested a variety of flap sub strates, Similar levels of PCNA stimulation occur on both a cleavage-sensit ive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y sub strate that exhibits upstream primer-independent cleavage. A pseudo-P subst rate with a sequence requiring an upstream primer for cleavage was not acti vated by PCNA, suggesting that PCNA does not compensate for substrate featu res that inhibit cleavage. A biotin streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not re store FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can l ower the K-m for FEN1 by 11-12-fold. Overall, our results indicate that aft er FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.