Single-molecule DNA digestion by lambda-exonuclease.
We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained lambda-DNA attached to optically trapped beads. The setup has been described previously (Dapprich and Nicklaus: Bioimaging 6:25-32, 1998) and works by observing the change in position of a trapped bead depending on its viscous drag force during motion. The drag force of a naked bead increases with each attached DNA molecule to a characteristic level that depends on the length and the number of DNAs per bead. A single undigested DNA molecule on a bead will remain stable for extended periods and exhibit a constant drag force in flow. If lambda-exonuclease is added, the drag force decreases from the level for one strand of DNA on a bead to that of a naked bead in about 45 min. This result indicates that the digestion of native lambda-DNA by lambda-exonuclease occurs at an average rate of approximately 15-20 Hz.
Dapprich
J
J
Praelux, Inc., Lawrenceville, New Jersey, USA. jdapprich@orchidbio.com
D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D014764:Viral Proteins
D004279:DNA, Viral; D014764:Viral Proteins; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda