Fluorometric assays for characterizing DNA helicases.

DNA helicases belong to an important class of motor proteins and are involved in almost all aspects of DNA metabolism. They hydrolyze NTP to translocate along ssDNA and unwind dsDNA by relying on chemical to physical energy transfer processes that are achieved via nucleotide-state-dependent conformational changes. For understanding the mechanisms by which helicases unwind DNA as well as their cellular functions, various properties of helicases need to be characterized. For these purposes, many assays have been developed, among which fluorometric assays are in the majority. Fluorometric assays are generally simple, direct and convenient to perform. Here we introduce several frequently used fluorometric assays for determining the basic properties of DNA helicases such as equilibrium ATP and DNA binding, kinetics of dissociation from DNA substrate and kinetics of DNA unwinding. Problems that may be encountered in experiments and possible ways to circumvent them are discussed. Copyright (c) 2010 Elsevier Inc. All rights reserved.