Single-molecule DNA digestion by lambda-exonuclease

Abstract

We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained λ-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 λ- 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 λ-DNA by λ-exonuclease occurs at an average rate of approximately 15-20 Hz.