Single-Molecule FRET Analysis of Replicative Helicases.

ABSTRACT

Over the recent years single-molecule fluorescence resonance energy transfer (smFRET) technique has proven to be one of the most powerful tools for revealing mechanistic insights into helicase activities. Here we describe details of single-molecule FRET assays for probing DNA unwinding activities as well as functional dynamics by replicative helicases in real time. The ability of smFRET to measure the behavior of biomolecules at a nanometer scale enabled us to address how the leading and lagging strand synthesis are coordinated during DNA replication, to resolve DNA unwinding steps of Bacteriophage T7 helicase, and to observe heterogeneous unwinding patterns modulated by the DNA binding domain of E1 helicase. These single-molecule FRET assays are generally applicable to other replicative and nonreplicative hexameric helicases.