RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis

Abstract

The DEAH-box ATP-dependent RHAU helicases specifically unfold RNA and DNA G-quadruplexes (G4s). However, it remains unclear how the RHAU's G4 unfolding activity is coupled to different stages of the ATPase cycle. Here, using a single-molecule manipulation approach, we show that binding of Drosophila RHAU stabilizes an intramolecularly folded parallel DNA G4 against mechanical unfolding in its nucleotide-free and in its AMP-PNP or ADP bound states, while it destabilizes the G4 when coupled to ATP hydrolysis. Importantly, our results show that the ADP·AlF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$_4^-$\end{document}-bound RHAU does not stabilize the G4. We also found that both a single-stranded 3′ DNA tail and the RSM domain of RHAU that binds specifically to the G4 structure, are dispensable for the stabilization of the G4, but both are required for G4 destabilization. Our study provides the first evidence that the unfolding kinetics of a G-quadruplex can be modulated by different nucleotide-bound states of the helicase.

The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism

“…This mechanism differs from a previous proposal that DHX36 uses a DEAD-box-like mechanism (26,42), which would imply disruption by direct binding of DHX36 to the G4 and the absence of DHX36 translocation. We find that the G4 disruption process is highly efficient, so much so that DHX36 binding to the 3′ extension limits the rate of disruption under subsaturating conditions.…”

“…A distinguishing feature of DEAH family helicases is that they typically require such an extension because the protein loads onto the single-stranded segment and then translocates 3′ to 5′ for helix unwinding. It was shown recently that DHX36 indeed requires a 3′ extension for efficient G4 disruption (27,42). However, little is known about the dependence of G4 disruption efficiency on the length of this 3′ extension, and the mechanistic origin of this requirement has not been systematically explored.…”

General and Target-Specific DExD/H RNA Helicases in Eukaryotic Translation Initiation

“…DHX36 does not discriminate between DNA and RNA substrates, as its contacts with the nucleic acid backbone are primarily with phosphates [123]. The drosophila DHX36 homolog has been shown to stabilize G4s in the absence of nucleotide or when bound to AMP-PNP or ADP, but resolves said structures in the presence of ATP [124]. The domains interacting with both the G4 structure and 3 tail are necessary for this destabilization [124].…”

“…The drosophila DHX36 homolog has been shown to stabilize G4s in the absence of nucleotide or when bound to AMP-PNP or ADP, but resolves said structures in the presence of ATP [124]. The domains interacting with both the G4 structure and 3 tail are necessary for this destabilization [124].…”

The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

“…For all experiments conducted in the presence of ATP, an additional regeneration system comprising 0.1 mg/mL pyruvate kinase (PK) from rabbit muscle and 2 mM phosphoenolpyruvate (PEP) were added. For all experiments conducted in the presence of ADP, the ADP solution used was treated with hexokinase and D-glucose to remove the contaminating ATP as described previously [35][36][37].…”