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J Biol Chem, Vol. 274, Issue 3, 1233-1239, January 15, 1999

Structural Changes Measured by X-ray Scattering from Human Flap Endonuclease-1 Complexed with Mg²⁺ and Flap DNA Substrate

Chang-Yub Kim, Binghui Shen, Min S. Park, and Glenn A. Olah^¶

From the  Life Sciences and ^¶ Chemical Science and Technology Divisions, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is essential in DNA replication and repair. FEN-1 has specific endonuclease activity for repairing nicked double-stranded DNA substrates that have the 5'-end of the nick expanded into a single-stranded tail, and it is involved in processing Okazaki fragments during DNA replication. Magnesium is a cofactor required for nuclease activity. We used small-angle 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 Mg²⁺ binding at the active site (Shen, B., Nolan, J. P., Sklar, L. A., and Park, M. S. (1996) J. Biol. Chem. 271, 9173-9176). Our data indicate that FEN-1 and the D181A mutant each have a radius of gyration of ~26 Å, and the effect of Mg²⁺ on the scattering from the proteins alone is insignificant. The 34-mer DNA fragment was constructed such that it readily forms a 5'-flap structure. The formation of the flap conformation of the DNA 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 absence of magnesium, the FEN-1·34-mer DNA flap complex has an R_g value of ~34 Å, whereas the D181A·34-mer DNA flap complex self-associates, suggesting that a significant protein conformational change occurs by 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 scattering profiles arising from magnesium activation of the FEN-1·34-mer DNA flap complex is consistent with the protein completely releasing the DNA substrate after cleavage.

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