G-quadruplex structures (G4s) form readily in DNA and RNA and play diverse roles in gene expression and other processes, and their inappropriate formation and stabilization are linked to human disease Show more
G-quadruplex structures (G4s) form readily in DNA and RNA and play diverse roles in gene expression and other processes, and their inappropriate formation and stabilization are linked to human diseases. G4s are inherently long-lived, such that their timely unfolding depends on a suite of DNA and RNA helicase proteins. Biochemical analysis of G4 binding and unfolding by individual helicase proteins is important for establishing their levels of activity, affinity, and specificity for G4s, including individual G4s of varying sequence and structure. Here we describe a set of simple, accessible methods in which electrophoretic mobility shift assays (EMSA) are used to measure the kinetics of G4 binding, dissociation, and unfolding by helicase proteins. We focus on practical considerations and the pitfalls that are most likely to arise when these methods are used to study the activities of helicases on G4s. Show less
DHX36 is a eukaryotic DEAH/RHA family helicase that disrupts G-quadruplex structures (G4s) with high specificity, contributing to regulatory roles of G4s. Here we used a DHX36 truncation to examine th Show more
DHX36 is a eukaryotic DEAH/RHA family helicase that disrupts G-quadruplex structures (G4s) with high specificity, contributing to regulatory roles of G4s. Here we used a DHX36 truncation to examine the roles of the 13-amino acid DHX36-specific motif (DSM) in DNA G4 recognition and disruption. We found that the DSM promotes G4 recognition and specificity by increasing the G4 binding rate of DHX36 without affecting the dissociation rate. Further, for most of the G4s measured, the DSM has little or no effect on the G4 disruption step by DHX36, implying that contacts with the G4 are maintained through the transition state for G4 disruption. This result suggests that partial disruption of the G4 from the 3' end is sufficient to reach the overall transition state for G4 disruption, while the DSM remains unperturbed at the 5' end. Interestingly, the DSM does not contribute to G4 binding kinetics or thermodynamics at low temperature, indicating a highly modular function. Together, our results animate recent DHX36 crystal structures, suggesting a model in which the DSM recruits G4s in a modular and flexible manner by contacting the 5' face early in binding, prior to rate-limiting capture and disruption of the G4 by the helicase core. Show less