Dataset Information

Identification of HELLS as a Novel DNA G-quadruplex Helicase

ABSTRACT: Guanine quadruplexes (G4s) are unique secondary structures of nucleic acids with functions in many biological processes. Understanding the biological functions of DNA G4s requires the knowledge about their recognitions by cellular proteins. Affinity pull-down coupled with LC-MS/MS analysis was previously employed to identify G4-binding proteins (G4BPs); the approach, however, has limitations in capturing weak and transient interactions, potentially leading to false-positives. Here, we developed a photoclick chemistry-based method, in combination with LC-MS/MS, for uncovering G4BPs. By incorporating a photoactivatable ortho-nitrobenzylamine (o-NBA) moiety into G4 DNA probes and employing UVA irradiation along with stringent washing, we identified 99 proteins enriched with G4 structures derived from the human telomere. We further validated the abilities of one of these proteins, HELLS, in binding and resolving G4 structures both in vitro and in chromatin. Together, we developed a photoclick chemistry-based approach for identifying novel G4BPs. Our work led to the discovery of new G4BPs, and uncovered novel functions of HELLS in recognizing and unwinding G4 structures in vitro and in cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Zi Gao

LAB HEAD: Zi Gao

PROVIDER: PXD051342 | Pride | 2026-02-02

REPOSITORIES: Pride

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Photoclick chemistry led to the identification of HELLS as a helicase for DNA G-quadruplexes.

Gao Zi Z Chong Yiewoon Y He Xiaomei X Yuan Jun J Wang Yinsheng Y

Nucleic acids research 20260101 3

Guanine quadruplexes (G4s) are unique secondary structures of nucleic acids with functions in many biological processes. Understanding the functions of DNA G4s requires knowledge about their recognition by cellular proteins. Here, we developed a method involving photoclick chemistry and LC-MS/MS-based quantitative proteomics for uncovering G4-binding proteins (G4BPs). By incorporating a photoactivatable ortho-nitrobenzylamine moiety into G4 DNA probes and employing UVA irradiation along with str ...[more]

PMID: 41569154

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Project description:DNA secondary structures are important for fundamental genome functions such as transcription and replication1. The G-quadruplex (G4) structural motif has been linked to gene regulation2,3 and genome instability4,5 and may be important to cancer development and other diseases6-8. Recently, ~700,000 discrete G4s have been observed in naked human single-stranded genomic DNA using G4-seq, a high-throughput sequencing technique that detects structural features in vitro.9 It is of vital importance to investigate G4 structures within an endogenous chromatin context, which until now remained elusive10,11. Herein, we address this via the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We identified ~10,000 endogenous G4 structures and show that G4s are predominantly seen in regulatory, nucleosome-depleted, chromatin regions. G4s were enriched in the promoters and 5âUTR regions of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Reorganization of the chromatin landscape using a histone deacetylase inhibitor, resulted in de novo G4 formation in new and more prominent regulatory, nucleosome-depleted regions associated with increased transcriptional output. Our findings suggest a striking relationship between promoter nucleosome-depleted regions, G4 formation and elevated transcriptional activity. Comparison between normal human epidermal keratinocytes and their immortalized counterparts revealed a ï¾7-fold greater G4 abundance in immortalized cells, of which 80 % were found in regulatory, nucleosome-depleted regions common to both cell types. Consequently, cells exhibiting more G4s displayed significantly increased transcriptional output and were more sensitive to growth inhibition by a small molecule G4 ligand. Overall, our results provide new mechanistic insights into where and when DNA adopts G4 structure in vivo. Our findings show for the first time that regulatory, nucleosome-depleted chromatin and transcriptional states predominantly shape the endogenous G4 DNA landscape. Two cell lines, treated with entinostat or untreated, analyzed to detect gene expression differences, presence of G-Qudruplexes and chromatin state. Each combination of conditions replicated in duplicates or triplicates.

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