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 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 approximately 26 A, 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 Io 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 Rg value of approximately 34 A, 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.
Kim
C Y
CY
Life Sciences, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Shen
B
B
Park
M S
MS
Olah
G A
GA
D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D045585:Flap Endonucleases; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D012542:Scattering, Radiation; D014965:X-Rays
D004247:DNA; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D008274:Magnesium