Gel-based assays for measuring DNA unwinding, annealing, and strand exchange

Abstract

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.