Investigating the Effects of Migrating Substrates, Proliferating Cell Nuclear Antigen and Arch Mutations on Human Flap Endonuclease-1 Activity

Flap endonucleases (FENs) in conjunction with proliferating cell nuclear antigen (PCNA) catalyse the removal of single-stranded 5'-DNA or RNA protrusions known as flaps that occur during DNA replication and repair. These bifurcated structures, which are the result of polymerase strand-displacement activity during lagging-strand DNA synthesis and repair, are likely migrating flaps in vivo due to sequence complementarity. In-vitro studies of FEN1 activity frequently use the optimal FEN1 substrate known as the static double-flap (DF), which consists of a single nucleotide 3' and a 5' flap of any length including zero. Moreover, the static DF substrates have 3' or 5' flaps that are not complementary to the template strand to reduce conformational variability. To understand better the mechanism of human FEN1 in a true biological context, nine migrating DF substrates were designed to determine the effect of increasing substrate-conformational complexity on the rates of FEN1 phosphate diester hydrolysis under multiple- and single-turnover conditions. The multiple-turnover analyses reveal that human FEN1 produces a single 5' flap product and nicked DNA even on equilibrating double flap substrates that have many potential conformers. This shows that hFEN1 action always recognises and hydrolyses a single conformer bearing a single 3'-nucleotide flap. The kcat/KM and kSTmax conditions showed generally that the rate of reaction was inversely proportional to the number of potential. Due to the decrease in hydrolytic efficiency with complex migrating flaps, whether PCNA could assist FEN1 in the hydrolysis of such substrates was investigated. Surprisingly, we found that PCNA did not stimulate the reactions of FEN1 with static or migrating double or single flaps. The implications of these results will be discussed.