Nucleotide specificity versus complex heterogeneity in exonuclease activity measurements.

A recent publication reported on measurements of Exonuclease I activity using a real-time fluorescence method that measures the time required by molecules of Exonuclease I to hydrolyze single-stranded DNA that was synthesized to have two fluorescently labeled nucleotides. The observed fluorescence-intensity curves were interpreted as a sign of strong heterogeneity of the activity of Exonuclease I. Here, I propose a different model, which assumes that Exonuclease I activity is nucleotide-dependent, and that a fluorescent label bound to a nucleotide significantly slows its cleavage rate. The presented model fits the observed data equally well, but can be used to make specific predictions upon observable sequence dependence of measured fluorescence-intensity curves.