Investigation of the hydrolysis of single DNA molecules using fluorescence video microscopy

We developed a method of studying the hydrolysis of single DNA molecules by exonuclease (exo) III using fluorescence video microscopy. A single DNA molecule immobilized at one end on a coverslip and labeled with a fluorescent bead at the other end confined the motion of a bead to a two-dimensional projected circular area determined by the contour length of DNA. The radius of this area decreased with time after the addition of exo III, Mg2+, or a single-stranded DNA-binding protein (SSB), which caused the single-stranded (ss) DNA to twist around itself. However, the radius was relatively constant over time in the absence of Mg2+, which is a cofactor in exo III activity, even when exo III and SSB were both present. These observations indicated that the decrease in the radius was due to hydrolysis of DNA by exo III. We then evaluated the rate of exo III hydrolysis of single DNA molecules by monitoring the decrease in the radius.