Nucleic Acids Research最新文献

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles in cancer and other genetic diseases. In a recent study, we discovered CBL0137, a curaxin ligand, to enhance cancer immunotherapy by inducing Z-DNA formation and activating the Z-DNA-binding protein ZBP1. However, the structural information on binding complexes between Z-DNA and CBL0137 ligand has not reported to date. Here we present the first high-resolution structure of the complex between a Z-DNA and a curaxin ligand CBL0137. This compound is observed to interact with the Z-DNA through π-stacking and zig-zag localization. Furthermore, we directly observe the complex in living human cells using in-cell 19F NMR for the first time. This structural information provides a platform for the design of topology-specific Z-DNA-targeting compounds and is valuable for the development of new potent anticancer drugs.

Nucleotide salvage is crucial for maintaining DNA replication when de novo nucleotide synthesis is limited, but this metabolic flexibility poses potential threats to genome stability. Salvage kinases phosphorylate nucleosides broadly, allowing for oxidized and alkylated 2'-deoxynucleosides as well as posttranscriptionally modified ribonucleosides to enter the 2'-deoxynucleoside triphosphate (dNTP) pool. The ensuing contamination of the dNTP pool and the subsequent incorporation of modified nucleotides into genomic DNA promote mutagenesis, induce replication stress, elicit double-strand breaks, and disrupt epigenetic signaling. Although only a small subset of modified nucleosides have been assessed for salvage and genomic incorporation, the scope of salvageable substrates is probably much wider, with significant implications in mutational burden, chromatin instability, and epigenetic regulation. This overlooked aspect of genome instability is especially relevant in biological contexts of high salvage activity or elevated nucleoside damage, including chronic inflammation, cancer, aging, and dietary/microbiome exposures. Emerging evidence links salvage metabolism to tumor progression, where incorporation of salvage-derived nucleotides may contribute to unexplainable mutational signatures detected in cancers, such as gastrointestinal cancer. Recognizing salvage as a hidden source of mutagenesis reshapes our understanding of genome instability and provides potential opportunities for disease prevention, diagnosis, and therapeutic intervention.

The cytoplasmic fate of messenger RNAs (mRNAs) is dictated by the balance of translation and mRNA degradation, governed in part by the 3' poly-adenosine tail and cytoplasmic poly(A)-binding proteins (PABPCs). Deadenylases remove poly(A) to initiate mRNA decay, while sequence-specific RNA-binding factors, including Pumilio proteins (PUM1 and PUM2), modulate these processes. We investigated how human PUM1&2 repress target mRNAs by accelerating their degradation. We found that the poly(A) tail plays a central role in PUM repression, dependent on the interplay of deadenylases and PABPCs. PUM-mediated repression requires the CCR4-NOT deadenylase but not the poly(A) nuclease. PUMs associate with and require PABPC1 and PABPC4 to repress. In the absence of PABPCs, both PUM targets and non-targets become unstable, bypassing PUM control. Increasing PABPC inhibits PUM activity in a concentration-dependent manner by stabilizing poly(A) mRNAs. The results support a Goldilocks principle, wherein PABPC abundance tunes the response of mRNAs to PUM-mediated repression through protection of poly(A) from deadenylation. We propose that this principle may apply to other poly(A) dependent regulatory factors. Variation of PABPC levels across tissues and development suggests physiological relevance for this mechanism.

Condensins are key regulators of chromosome architecture and have emerging functions in DNA repair that are understudied. Here, we show that combined depletion of Condensin I and II in cell lines of normal and tumor origin selectively impairs DNA double-strand break (DSB) repair and the checkpoint response (DDR) specifically in the G2 phase of the cell cycle, with no detectable effects in G1 or S phase. Condensin knockdown increased cellular radiosensitivity and delayed in G2-phase, but not in asynchronous cells, the resolution of γH2AX and 53BP1 foci, indicating G2-specific defects in DSB repair. Mechanistically, condensin loss suppressed DNA end-resection and resection-dependent repair pathways, including homologous recombination (HR), single-strand annealing (SSA), and alternative end-joining (alt-EJ), but failed to significantly alter classical non-homologous end-joining (c-NHEJ). Reduced RAD51 and RPA70 foci formation in G2 confirmed inhibition of HR and DNA end resection. The G2 checkpoint was also compromised. Cytogenetic analysis revealed inhibition of chromosome break repair and visible chromatin decondensation, suggesting that condensins function to maintain an appropriate chromatin state for efficient DSB repair in G2-phase. These results identify for the first time condensins as G2 phase-specific regulators of genome stability by fine-tuning HR and other resection-dependent DSB repair pathways.

Anchoring of a chromatin remodeler complex by long non-coding RNAs (lncRNAs) is a frequently utilized mechanism for lncRNAs to regulate gene expression. Hypoxia is a microenvironmental condition that plays a crucial role in promoting tumor progression. We previously identified a hypoxia-inducible lncRNA, RP11-390F4.3, that regulates epithelial-mesenchymal transition (EMT) without a delineated mechanism. Here, we show that the lncRNA RP11-390F4.3 (renamed MAHAC: MAintenance of Histone ACetylation) specifically induces histone H4 lysine 5 acetylation (H4K5ac) mark and promotes the deposition of H4K5ac mark on the promoters of EMT transcription factors. MAHAC scaffolds the ILF3/NF90-ILF2-CBP complex, which is co-localized with the members of the complex inside the nucleus under hypoxia. The minimal MAHAC region (nt 686-741) required for scaffolding the complex was mapped, and it induces allosteric activation of H4K5ac in in vitro histone acetyltransferase assay. This minimal MAHAC region is essential for hypoxia-induced EMT, migration, invasion, and H4K5ac activation. These findings demonstrate that hypoxia-induced MAHAC represents an unexplored allosteric regulator of H4K5ac that activates EMT and induces tumor progression.

Archaea, a domain of microorganisms found in diverse environments, including the human microbiome, represent the closest known prokaryotic relatives of eukaryotes. This phylogenetic proximity positions them as a relevant model for investigating the evolutionary origins of nucleic acid secondary structures such as G-quadruplexes (G4s) which play regulatory roles in transcription and replication. Although G4s have been extensively studied in eukaryotes, their presence and function in archaea remain poorly characterized. In this study, a genome-wide analysis of the halophilic archaeon Haloferax volcanii identified over 5800 potential G4-forming sequences. Biophysical validation confirmed that many of these sequences adopt stable G4 conformations in vitro. Using G4-specific detection tools and super-resolution microscopy, G4 structures were visualized in vivo in both DNA and RNA across multiple growth phases. Comparable findings were observed in the thermophilic archaeon Thermococcus barophilus. Functional analysis using helicase-deficient H. volcanii strains further identified candidate enzymes involved in G4 resolution. These results establish H. volcanii as a tractable archaeal model for G4 biology.

In human cells, the Nuclear EXosome Targeting (NEXT) and Poly(A) tail eXosome Targeting (PAXT) adaptors direct the nuclear exosome to degrade prematurely terminated RNA Polymerase II (Pol II) transcripts, ensuring nuclear RNA quality control. How these adaptors interact with transcription termination machineries remains largely unclear. Here, we leveraged in silico structure predictions of protein complexes to identify and model previously unreported interactions of NEXT- and PAXT-associated components with two transcription termination and processing machineries, the Integrator and Cleavage and Polyadenylation (CPA) complexes. Our computational models were validated through complementary in vitro biochemical approaches and single-particle cryo-EM analyses. We show that the ZC3H18 protein uses two different domains to directly recognize the INTS9/11 endonuclease module of Integrator and the mammalian Polyadenylation Specificity Factor (mPSF), a core CPA component. In turn, ZC3H18 can directly bind the scaffolding subunits of NEXT and PAXT via mutually exclusive interactions. Furthermore, we provide evidence that accessory PAXT components can be directly integrated with the mPSF core, establishing configurations that are mutually exclusive with those of canonical CPA subunits. These findings reveal a versatile interaction network capable of forming alternative structural frameworks linking transcription termination with nuclear RNA quality control.

DNMT1 is a methyltransferase that restores 5-methylcytidine marks on newly replicated DNA and is required for maintaining epigenetic inheritance. Using Halo-tagged DNMT1 and highly inclined thin illumination (HiLo) microscopy, we show that DNMT1 mobility in living human cells changes under a variety of conditions. DNMT1 molecules become increasingly bound to chromatin in the S phase of the cell cycle, but surprisingly only ∼ 12% chromatin-bound DNMT1 is sufficient to maintain DNA methylation. Upon treatment with small molecule inhibitors, GSK-3484862 (GSK), 5-azacytidine (5-azaC) and decitabine (5-aza-deoxyC), in vivo DNMT1 dynamics are greatly altered. Unexpectedly, treatment of cells with GSK, a non-covalent inhibitor, causes binding of DNMT1 to chromatin similar to that observed upon treatment with 5-azaC and decitabine, covalent inhibitors. 5-azaC inhibition of DNMT1 dynamics occurs during the S phase of the cell cycle. Unexpectedly, mutations in the disordered, Asp- and Glu-rich N-terminal region of DNMT1 dramatically decrease its mobility and increase chromatin binding. Collectively, our work using live cell single molecule imaging quantifies the molecular dynamics of DNMT1 and how this relates to its function under physiological conditions and upon drug treatment. Understanding the dynamics of DNMT1 in vivo provides a framework for developing better therapeutics that target DNMT1.

The ribosomal RNA gene cluster (rDNA) in Saccharomyces cerevisiae consists of about 150 tandem copies, making it a fragile site prone to copy number changes through recombination among the repeat. While extensive research has been conducted to understand the mechanisms for rDNA stability maintenance, the relationship between the stability maintenance of rDNA and other genomic regions remains unclear. In this study, we identified a mutant, sic1, that exhibited instability in both rDNA and chromosome IV (chr.IV). We revealed that Ty element-mediated ectopic recombination leads to partial duplication and elongation of chr.IV. Furthermore, we found that rDNA instability is caused by an increased SIR4 gene dosage resulting from this partial duplication. These findings suggest a link between the stability of rDNA and other genomic regions.