DNA unwinding assay using streptavidin-bound oligonucleotides

An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'→ 5' and 5'→ 3' helicases was developed.It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques.Helicases play important roles in DNA and RNA metabolic processes such as replication, recombination, repair, transcription and translation [1]. The enzyme catalyzes the unwinding of duplex nucleic acid, utilizing the energy derived from nucleoside triphosphate hydrolysis to translocate along one strand of the duplex and to displace the complementary strand. Thus, most helicases have a specific polarity. They move along the strand either in the 3'→ 5' or 5'→ 3' direction. Some helicases, however, are bipolar and can move in either direction (e.g. [2]). Thus, one of the first properties to be determined for a new helicase is its directionality.To date, several different substrates have been used to determine helicase directionality. One such substrate is schematically shown in Figure 1A[3]. It is made by annealing a 5' 32P-labled complementary strand to a blunt end restriction enzyme site on a single-stranded plasmid DNA (e.g. M13 or φX174), followed by 32P labeling of the 3' end using 32P-labled dNTP and DNA polymerase. The labeled substrate is then digested with the restriction enzyme. The result is a linear molecule with a large internal region of ssDNA where a helicase can assemble, and two 32P-labled duplex regions of different lengths. Depending on enzyme polarity, only