Fluorescence tools to measure helicase activity in real time

Methods are described to show how different fluorescent labeling strategies can be used to probe various aspects of the helicase mechanism. Fluorophores on the adenine nucleotide, the DNA or the helicase can modify the activity of the system to a greater or lesser extent. Reagentless biosensors, binding proteins that are labeled with a fluorophore, target products of the helicase reaction, namely ADP, inorganic phosphate or single-stranded DNA, and can be used to measure rates of product formation with little interference to the system. Protocols are described to examine ATP usage and translocation speeds and also to investigate details of the ATP hydrolysis cycle. The methods are described in terms of PcrA, a bacterial DNA helicase that moves in single base steps along either single-stranded or double-stranded DNA, hydrolyzing one ATP per base moved.