Polymerase η Recruits DHX9 Helicase to Promote Replication across Guanine Quadruplex Structures

Tang, Feng; Wang, Yinan; Gao, Zi; Guo, Shiyuan; Wang, Yinsheng

doi:10.1021/jacs.2c05312

Cited by 18 publications

(11 citation statements)

References 37 publications

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“…To further examine the roles of phase separation in stabilizing G4 structures in cells, we performed BG4-ChIPseq experiment for U2OS cells that were untreated or treated with 1.5% 1,6-HD for 2 min. 30,31 Our results from two biological replicates revealed a substantially diminished number of BG4 ChIP-seq peaks, namely, from 3507 peaks in control cells to 2269 peaks in 1,6-HD-treated cells. The majority of the peaks (58.9 and 61% in control and 1,6-HDtreated cells, respectively) are located in promoter regions (Figure 3A), which is in agreement with the previously reported results.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…G4-ChIP sequencing was performed using the custom-purified BG4 antibody and conducted as previously described with minor modifications . DNA was fragmented following the protocol described previously . Briefly, chromatin samples were diluted in a blocking buffer containing 25 mM HEPES (pH 7.5), 10.5 mM NaCl, 110 mM KCl, 1 mM MgCl 2 , and 1% BSA and treated with RNase A.…”

Section: Methodsmentioning

confidence: 99%

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Phase Separation Modulates the Formation and Stabilities of DNA Guanine Quadruplex

Gao

Yuan

et al. 2023

JACS Au

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In the presence of monovalent alkali metal ions, Grich DNA sequences containing four runs of contiguous guanines can fold into G-quadruplex (G4) structures. Recent studies showed that these structures are located in critical regions of the human genome and assume important functions in many essential DNA metabolic processes, including replication, transcription, and repair. However, not all potential G4-forming sequences are actually folded into G4 structures in cells, where G4 structures are known to be dynamic and modulated by G4-binding proteins as well as helicases. It remains unclear whether there are other factors influencing the formation and stability of G4 structures in cells. Herein, we showed that DNA G4s can undergo phase separation in vitro. In addition, immunofluorescence microscopy and ChIPseq experiments with the use of BG4, a G4 structure-specific antibody, revealed that disruption of phase separation could result in global destabilization of G4 structures in cells. Together, our work revealed phase separation as a new determinant in modulating the formation and stability of G4 structures in human cells.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Phase Separation Modulates the Formation and Stabilities of DNA Guanine Quadruplex

Gao

Yuan

et al. 2023

JACS Au

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“…It can be anticipated that the pathogenic RFC1 harboring hundreds to thousands of AAGGG repeats may exert a more detrimental function to disease pathogenesis. Recently, the importance of G4-resolvases and G4-unfolding ligands has been increasingly recognized for neurodegenerative disease therapy ( 66–69 ). For instance, the Cockayne Syndrome B protein and DEAH-Box helicase 9 could resolve DNA G4 structures to restore normal cellular activities ( 66 , 67 ).…”

Section: Discussionmentioning

confidence: 99%

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS

Wang,

Yan

et al. 2024

Nucleic Acids Research

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An expansion of AAGGG pentanucleotide repeats in the replication factor C subunit 1 (RFC1) gene is the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and it also links to several other neurodegenerative diseases including the Parkinson's disease. However, the pathogenic mechanism of RFC1 AAGGG repeat expansion remains enigmatic. Here, we report that the pathogenic RFC1 AAGGG repeats form DNA and RNA parallel G-quadruplex (G4) structures that play a role in impairing biological processes. We determine the first high-resolution nuclear magnetic resonance (NMR) structure of a bimolecular parallel G4 formed by d(AAGGG)2AA and reveal how AAGGG repeats fold into a higher-order structure composed of three G-tetrad layers, and further demonstrate the formation of intramolecular G4s in longer DNA and RNA repeats. The pathogenic AAGGG repeats, but not the nonpathogenic AAAAG repeats, form G4 structures to stall DNA replication and reduce gene expression via impairing the translation process in a repeat-length-dependent manner. Our results provide an unprecedented structural basis for understanding the pathogenic mechanism of AAGGG repeat expansion associated with CANVAS. In addition, the high-resolution structures resolved in this study will facilitate rational design of small-molecule ligands and helicases targeting G4s formed by AAGGG repeats for therapeutic interventions.

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“…The DNA G4 formed in RFC1 PRE appears to elicit detrimental effects on several key biological processes, and thus may be a potential therapeutic target. It has been recently reported that proteins or helicases, such as the Cockayne Syndrome B protein and DEAH-Box helicase 9, could resolve pathogenic DNA G4 structures to restore normal cellular function 46,47 . The unprecedented high-resolution structures of G4 formed by AAGGG repeats will facilitate the discovery of helicase and small-molecule ligands to resolve the culpable G4 structure in RFC1 PRE for therapeutic intervention.…”

Section: Discussionmentioning

confidence: 99%

RFC1AAGGG pentanucleotide repeats form parallel G-quadruplex: structural implications for aberrant molecular cascades in CANVAS

Wang,

Yan

et al. 2023

Preprint

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A pentanucleotide repeat expansion (PRE) of (AAGGG)n in the replication factor C subunit 1 (RFC1) gene is recently identified as the genetic cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS), and also linked to several other neurodegenerative disorders including the Parkinson's disease. However, the molecular mechanism by which RFC1 PRE drives pathology remains poorly understood. Here, for the first time we discovered and determined the high-resolution structures of parallel G-quadruplex formed by AAGGG repeats within the pathogenic RFC1 PRE, revealing an intriguing conformational plasticity at the 3'-termi that allows stacking of multiple G4s. We further identify a molecular mechanism by which the DNA G4 in RFC1 PRE impedes polymerase processivity leading to replication stalling and transcription inhibition in vitro in a repeat-length-dependent manner, and the transcription inhibition could partially contribute to a reduced gene expression in cells. Our results demonstrate that the DNA G-quadruplex of RFC1 PRE participate in aberrant molecular cascades, and provide an unprecedented high-resolution structural target to discover helicases and ligands that resolve the pathogenic G4 for therapeutic intervention.

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Polymerase η Recruits DHX9 Helicase to Promote Replication across Guanine Quadruplex Structures

Cited by 18 publications

References 37 publications

Phase Separation Modulates the Formation and Stabilities of DNA Guanine Quadruplex

Phase Separation Modulates the Formation and Stabilities of DNA Guanine Quadruplex

Structural investigation of pathogenic RFC1 AAGGG pentanucleotide repeats reveals a role of G-quadruplex in dysregulated gene expression in CANVAS

RFC1AAGGG pentanucleotide repeats form parallel G-quadruplex: structural implications for aberrant molecular cascades in CANVAS

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