Essential amino acids for substrate binding and catalysis of human flap endonuclease 1.
Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity.
Shen
B
B
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Nolan
J P
JP
Sklar
L A
LA
Park
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004256:DNA Polymerase I / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D045585:Flap Endonucleases; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004256:DNA Polymerase I; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human