The 3'-flap pocket of human flap endonuclease 1 is critical for substrate binding and catalysis.
Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3'-extrahelical nucleotide (3'-flap) binding pocket. When presented with 5'-flap substrates having a 3'-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3'-flap on substrates reduced Km and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3'-flap, and the absence of a 3'-flap from a 5'-flap substrate was more detrimental to hFEN1 activity than removal of the 5'-flap or introduction of a hairpin into the 5'-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3'-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5'-phosphorylated product. Together the results indicate that the presence of a 3'-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.
Finger
L David
LD
Division of Radiation Biology, Duarte, California 91010.
Blanchard
M Suzette
MS
Biostatistics, Duarte, California 91010.
Theimer
Carla A
CA
Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222.
Sengerová
Blanka
B
Centre for Chemical Biology, Department of Chemistry Krebs Institute, University of Sheffield, Sheffield, S3 7HF, United Kingdom.
Singh
Purnima
P
Division of Radiation Biology, Duarte, California 91010.
Chavez
Valerie
V
Division of Radiation Biology, Duarte, California 91010; Graduate School of Biological Sciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010.
Liu
Fei
F
Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222.
Grasby
Jane A
JA
Centre for Chemical Biology, Department of Chemistry Krebs Institute, University of Sheffield, Sheffield, S3 7HF, United Kingdom.
Shen
Binghui
B
Division of Radiation Biology, Duarte, California 91010.
D001483:Base Sequence; D002384:Catalysis; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D011189:Potassium Chloride / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013816:Thermodynamics
D011189:Potassium Chloride; D045585:Flap Endonucleases