Partial functional deficiency of E160D flap endonuclease-1 mutant in vitroand in vivo is due to defective cleavage of DNA substrates

To assess the roles of the active site residues Glu(160) and Asp(181) of hu man FEN-1 nuclease in binding and catalysis of the flap DNA substrate and i n vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the F EN-1 gene. The mutants were then expressed in Escherichia coil and purified using a His-tag. Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exceptio n of an E160D mutant. This mutant retained wild type-like binding ability, specificity, and partial catalytic activity. Detailed steady state and pre- steady state kinetic analysis revealed that the functional deficiency of th is mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the bi ological functions of the homologous yeast gene, RAD27, and reverses the hy per-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.