Bloom’s syndrome (BS) is a rare human autosomal recessive disorder characterized by a strong predisposition to a wide range of cancers commonly affecting the general population. Understanding the functioning mechanism of the BLM protein may provide the opportunity to develop new effective therapy strategies. In this work, we studied the DNA unwinding kinetic mechanism of the helicase core of the BLM protein using various stopped-flow assays. We show that the helicase core of BLM unwinds duplex DNA as monomers even under conditions strongly favoring oligomerization. An unwinding rate of ∼20 steps per second and a step size of 1 bp have been determined. We have observed that the helicase has a very low processivity. From dissociation and inhibition experiments, we have found that during its ATP hydrolysis cycle in DNA unwinding the helicase tends to dissociate from the DNA substrate in the ADP state. The experimental results imply that the BLM helicase core may unwind duplex DNA in an inchworm manner.

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.