Gel-Based Assays for Measuring DNA Unwinding, Annealing, and Strand Exchange

Efficient replication and repair of the genome requires a multitude of protein-DNA transactions. These interactions can result in a variety of consequences for DNA such as the unwinding of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), the annealing of complementary ssDNAs, or the exchange of ssDNA with one strand of a dsDNA duplex. Some DNA helicases possess all three activities, but many DNA-interacting proteins can also catalyze one or more of these reactions. Assays that quantify these activities are an important first step in characterizing these protein-DNA interactions in vitro. Here, we describe methods for the formation of dsDNA substrates and the assays that can be used to biochemically characterize proteins that can unwind, anneal, and/or exchange DNA strands.