Kinetic analysis of human flap endonuclease-1 by flow cytometry.
Human flap endonuclease-1 (FEN-1) is a structure-specific endonuclease and exonuclease which is essential for DNA replication and repair. We have cloned a human FEN-1 gene, overexpressed it in Escherichia coli, purified the recombinant protein to near homogeneity, and characterized its cleavage of a flap DNA structure using a novel analytical approach based on flow cytometry. With this approach, we were able to measure continuously the kinetics of DNA cleavage by FEN-1 and to separate experimentally the binding and catalysis functions of the enzyme. When the reaction was initiated by the addition of FEN-1, the cleavage kinetics were dependent on enzyme concentration and appeared to saturate at high concentrations. When enzyme and substrate were preincubated in the presence of EDTA and the reaction initiated by the addition of Mg2+, rapid kinetic flow cytometry measurements showed that cleavage is fast (t1/2 approximately 6 s, k = 0.10 s-1). Using the single-turnover kinetics as a measure of the amount of enzyme-substrate complex present, we estimated the Kd for the FEN-1-flap DNA substrate to be 7.5 nM in the absence of Mg2+ and the rate constant for dissociation of the enzyme-substrate complex to be 0.07 s-1. Computer fitting of the experimental data to a kinetic model confirms these estimates for the individual steps and suggests some interesting features of enzymology using a surface-bound substrate.
Nolan
J P
JP
Life Sciences Division and National Flow Cytometry Resource, Los Alamos National Laboratory, New Mexico 87545, USA.
Shen
B
B
Park
M S
MS
Sklar
L A
LA
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D045585:Flap Endonucleases; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008863:Microspheres; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D004492:Edetic Acid; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D008274:Magnesium