Helicase Unwinding at the Replication Fork

Ring-shaped hexameric helicases play an essential role of double-stranded DNA unwinding during genome replication. The NTPase-powered unwinding activity of the hexameric helicases is required both for replication initiation and fork progression. We describe ensemble biophysical methods to measure the unwinding activity of ring-shaped helicases during fork progression using the T7 bacteriophage replicative helicase gp4A′ as a model enzyme. These assays provide insights into the stepping mechanism of translocation, active or passive mechanism of unwinding, and regulation by associated proteins such as single strand DNA binding protein, DNA polymerase, and primase enzymes.