Partial functional deficiency of E160D flap endonuclease-1 mutant in vitro and in vivo is due to defective cleavage of DNA substrates.
To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene. The mutants were then expressed in Escherichia coli 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 exception 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 this mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.
Frank
G
G
Department of Cell and Tumor Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010, USA.
Qiu
J
J
Somsouk
M
M
Weng
Y
Y
Somsouk
L
L
Nolan
J P
JP
Shen
B
B
D001483:Base Sequence; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D045585:Flap Endonucleases; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D008741:Methyl Methanesulfonate; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; C468323:RAD27 protein, S cerevisiae