Digestion of individual DNA molecules by lambda-exonuclease at liquid-solid interface.
Enzyme digestion of single DNA molecules was directly observed in real time by dual-color total internal reflection fluorescence microscopy (TIRFM). Individual lambda-DNA molecules labeled with the fluorescent dye, YOYO-1, were stretched in a laminar flow stream and immobilized on a bare fused-silica prism surface based on hydrophobic and electrostatic interactions. Enzyme digestion was initiated by the influx of lambda-exonuclease enzyme via capillary force. When the dye : bp ratio was higher than 1 : 20, the exact digestion rate could not be measured because of induced photocleavage of the DNA molecules. At a dye : bp ratio of 1 : 50, shortening of the DNA strand was recorded in real time. Unlike previous studies, the length-based digestion rate of lambda-exonuclease showed 3 distinct values in the range of 0.173(+/-0.024) to 0.462(+/-0.152) microm s(-1) at 37 degrees C. That is, different enzyme molecules exhibit different digestion dynamics. Digestion was also monitored based on the decrease in fluorescence intensity, but uncertainties were much larger due to the distance dependent excitation intensity in the TIRF mode.
Kang
Seong Ho
SH
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Lee
Seungah
S
Yeung
Edward S
ES
D010582:Bacteriophage lambda / Q000201:enzymology*; D001583:Benzoxazoles / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D011806:Quinolinium Compounds / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D012822:Silicon Dioxide; D004247:DNA; D005090:Exodeoxyribonucleases