Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36

Abstract

Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than what most polymerases can exert1,2. Eukaryotic cells host numerous helicases capable of unfolding G-quadruplexes3. The molecular basis for helicase recognition and unfolding of G-quadruplexes remains poorly understood. DHX36 (RHAU, G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with uniquely high affinity4,5,6, is consistently found bound to G-quadruplexes in cells7,8, and is a major source of G-quadruplex unfolding activity in HeLa cell lysates6. DHX36 is a multi-functional helicase implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, hematopoiesis, and embryogenesis in mice9,10,11,12. Here, we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3’ single-stranded (ssDNA) segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that together with the OB-fold-like subdomain selectively binds parallel G-quadruplexes. Comparison with our unliganded and ATP-analog-bound DHX36 structures, together with single-molecule FRET analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core, which by pulling on its ssDNA tail, drive G-quadruplex unfolding by one residue at a time.

DDX5 helicase resolves G-quadruplex and is involved in MYC gene transcriptional activation

“…BLM and WRN, members of RecQ family helicases, were the first human helicases reported to resolve G4 DNA in human telomeres (63) and these enzymes function in a 3′→5′ manner (34). The DEAH-box helicase DHX9 and DHX36 (RHAU) were also shown to unfold both RNA and DNA G4 3′→5′ (64)(65)(66). Both DHX9 and DHX36 require a 3′ singlestranded region, which is sequence-specific, for loading and G4-unfolding (64)(65)(66).…”

“…The DEAH-box helicase DHX9 and DHX36 (RHAU) were also shown to unfold both RNA and DNA G4 3′→5′ (64)(65)(66). Both DHX9 and DHX36 require a 3′ singlestranded region, which is sequence-specific, for loading and G4-unfolding (64)(65)(66). All of the reported active G4-helicases are directional/processive and require a single-stranded tail at either the 3′ or 5′ end for loading and G4 unfolding (34).…”

Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43 acts on the catalytic snRNA U6

“…Consistent with its membership in the DExH family of ATPases, Prp43p unwinds duplexes specifically with a 3' overhang (He et al 2017), implying translocation along RNA in the 3' to 5' direction (Jankowsky 2011). Crystal structures of Prp43, Prp22, as well as of the DEAH ATPase MLE, bound to RNA and non-hydrolyzable ATP provided the first structural insights into the mechanism of translocation by the DEAH family members (He et al 2010;Prabu et al 2015;Tauchert et al 2017;He et al 2017;Hamann et al 2019;Chen et al 2018). The two RecA domains bind an RNA stack of four nucleotides that is bookended at the 5' end by an elongated beta-hairpin, as in the viral SF2 ATPase NS3 (Luo et al 2008;Gu and Rice 2010;Appleby et al 2011), and at the 3' end by a DEAH-specific elongation of motif Ib, which includes a residue that is required for RNA unwinding and mutated in human Prp43p in acute myeloid leukemias (Faber et al 2016).…”

A 5’ UTR G-quadruplex controls localisation and translation of a potassium leak channel mRNA

“…DHX36 (also known as RHAU and G4R1) is a G4-specific helicase and has been shown to bind preferentially to G4 and G-rich sequences on over 4500 mRNA transcripts, preventing accumulation of translationally repressed mRNAs (53). Chen et al recently co-crystallised DHX36 bound to the c-Myc G4, providing valuable insight into the mechanisms underpinning helicase mediated G4 unwinding (75). A mechanism of non-processive unwinding of G4es has been proposed, suggesting a preferential ATPindependent mechanism for DHX36 unwinding of RNA G4s, differing to that for DNA G4s (76,77).…”