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Nucleotide Specificity versus Complex Heterogeneity in Exonuclease Activity Measurements

Institut für Neurowissenschaft und Biophysik 1, Forschungszentrum Jülich, Jülich, Germany

Correspondence: Address reprint requests to Jörg Enderlein, Institut für Neurowissenschaft und Biophysik 1, Forschungszentrum Jülich, Jülich, Germany. E-mail: j.enderlein{at}fz-juelich.de.

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.