Fluorescent biosensors to investigate helicase activity

Abstract

ATP-driven translocation of helicases along DNA can be assayed in several ways. Reagentless biosensors, based on fluorophore-protein adducts, provide convenient ways for real-time assays of both the separation of dsDNA and the hydrolysis of ATP. Single-stranded DNA can be assayed using a modified single-stranded DNA-binding protein (SSB), and phosphate production during ATP hydrolysis can be measured by a modified phosphate-binding protein. Advantages and limitations of these approaches are compared with those of other types of measurements.