Structural changes measured by x-ray scattering from human flap endonuclease-1 complexed with Mg2+ and flap DNA substrate

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific end onuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA sub strates that have the 5'-end of the nick expanded into a single-stranded ta il, and it is involved in processing Okazaki fragments during DNA replicati on. Magnesium is a cofactor required for nuclease activity. We used small-a ngle x-ray scattering to obtain global structural information pertinent to nuclease activity from FEN-1, the D181A mutant, the wild-type FEN-1.34-mer DNA flap complex, and the D181A 34-mer DNA flap complex. The D181A mutant, which has Asp-181 replaced by Ala, selectively binds to the flap structure, but has lost its cleaving activity. Asp-181 is thought to be involved in M g2+ binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A. and Pa rk, M. S. (1996) J. Biol. Chem. 271, 9173-9176). Our data indicate that PEN -1 and the D181A mutant each have a radius of gyration of similar to 26 Ang strom and the effect of Mg2+ on the scattering from the proteins alone is i nsignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5'-flap structure. The formation of the flap conformation of the DN A substrate was evident by both the extrapolated I-o scattering and radius of gyration and was supported by NMR spectrum and nuclease assays. In the a bsence of magnesium, the FEN-1.34-mer DNA flap complex has an R-g value of similar to 34 Angstrom whereas the D181A 34-mer DNA flap complex self-assoc iates, suggesting that a significant protein conformational change occurs b y addition of the flap DNA substrate and that Asp-181 is crucial for proper binding of the protein to the DNA substrate. A time course change in the s cattering profiles arising from magnesium activation of the FEN-1.34-mer DN A flap complex is consistent with the protein completely releasing the DNA substrate after cleavage.