Abstract
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.
Highlights
G-quadruplexes (G4s) form by self-assembly of four guanosine tracts within one or more DNA or RNA strands
To probe the roles of the DHX36-specific motif (DSM), we first measured the steadystate kinetics of DNA G4 disruption by DHX36WT and DHX36ΔDSM
Using a five-quartet G4 with a 3’ extension of 15 adenosine nucleotides (5G-A15; Table S1), we found that DHX36ΔDSM disrupts subsaturating concentrations of this G4 with a secondorder rate constant of (7.3 ± 0.7) × 105 M−1 min−1 (Figure 2B)
Summary
G-quadruplexes (G4s) form by self-assembly of four guanosine tracts within one or more DNA or RNA strands. Putative G4-forming sequences are conserved and overrepresented in the promoter regions of human proto-oncogenes while underrepresented in tumor suppressor genes, suggesting key roles of G4s in regulation of gene expression and cancer (Hänsel-Hertsch et al 2017). These sequences are overrepresented in the 5’ and 3’ untranslated regions of mRNA, and RNA G4s have been suggested to play regulatory roles in translation and mRNA lifetime (Bugaut and Balasubramanian 2012; Beaudoin and Perreault 2013; Sauer et al 2019). One helicase of particular interest, DHX36 ( known as RHAU), is an ATP-dependent G4 resolvase that is highly expressed in mammals, essential for mouse development, and constitutes the bulk of the G4-resolving activity in human cell lysate (Creacy et al 2008; Lai et al 2012; Vaughn et al 2005; Yangyuoru et al 2018)
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