Nucleic Acids Research最新文献

The expanded backbone chemistries of xeno-nucleic acids (XNAs) hold significant promise for emerging areas of synthetic biology and nanomaterials, but metal-mediated XNA interactions remain largely unexplored. Here, we use a combination of circular dichroism spectroscopy and mass spectrometry to show that XNAs can form Ag+-mediated duplex structures resembling their DNA counterparts. XNAs with a range of different backbone compositions are found to stabilize photoluminescent silver nanoclusters with spectral properties that can be tuned based on their respective backbone chemistry. The resistance of silver nanoclusters to nuclease digestion is also compared for DNA and XNAs. These results show that XNA backbone chemistry provides a new tool beyond nucleobase sequence for controlling and expanding the properties of nucleic acid-stabilized silver nanoclusters and metal-mediated DNA duplexes.

DNA ends are frequently damaged during the formation of DNA double-strand breaks (DSBs). These ends must be repaired to enable ligation during non-homologous end joining (NHEJ). NHEJ uses several end processing factors to repair DNA ends within the short-range synaptic complex (SRC), including Polymerase λ (Pol λ) which performs gap fill-in. Pol λ possesses a Ku Binding Motif (KBM) within its BRCT domain that interacts with Ku and recruits it to the SRC. Here, we show that in addition to its role in recruitment, Ku also stimulates Pol λ polymerase activity at DSBs. Using a structural prediction approach and biochemical assays, we identify and characterize an autoinhibitory intramolecular interaction between the N-terminal BRCT and C-terminal catalytic domains of Pol λ. Furthermore, single-molecule approaches reveal that Ku increases both the binding rate of Pol λ to primer-template DNA and the rate of nucleotide incorporation, demonstrating that Ku releases Pol λ autoinhibition and stimulates its polymerase activity within the SRC during NHEJ. Combined, these data highlight how intricate protein-protein interactions within the SRC complex are critical to regulate end-processing and maximize the fidelity of DSB repair.

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.

Chronic hepatitis B virus (HBV) persistence relies on the chromatin plasticity of covalently closed circular DNA (cccDNA), a viral minichromosome resistant to current therapies. Using proximity labeling (TurboID-dCas9), ChIP-seq and DNA pull-down assays, we identified SMARCC2-a BAF scaffolding subunit-bound to cccDNA enhancer-promoter regions (EnhⅠ/XP, CP/EnhII), where it sustains nucleosome-depleted regions (NDRs) and recruits RNA polymerase II. Genetic or pharmacological BAF inhibition compacted cccDNA chromatin, reduced histone acetylation (AcH3/AcH4), and enhanced SMC5/6-mediated silencing to suppress transcription, with the BAF ATPase inhibitor FHT-2344 reducing serum HBV DNA by 50% (P <.05) and intrahepatic HBV RNA by 70% (P <.01) without cccDNA loss, indicating epigenetic silencing. Mechanistically, BAF maintains NDRs by counteracting nucleosome retention and recruiting host transcription factors such as HNF4α. This work concludes that BAF safeguards cccDNA chromatin plasticity to enable viral persistence, and targeting BAF (e.g. FHT-2344) epigenetically silences cccDNA, offering a novel strategy for functional cure.

Despite the many advances in single cell genomics, detecting structural rearrangements in single cells, particularly error-free sister-chromatid exchanges, remains challenging. Here we describe sci-L3-Strand-seq, a combinatorial indexing method with linear amplification for DNA template strand sequencing that cost-effectively scales to millions of single cells, as a platform for mapping mitotic crossover (CO) and resulting genome instability events. We provide a computational framework to fully leverage the throughput, as well as the relatively sparse but multifaceted genotype information within each cell that includes strandedness, digital counting of copy numbers, and haplotype-aware chromosome segmentation, to systematically distinguish seven possible types of mitotic CO outcomes. We showcase the power of sci-L3-Strand-seq by quantifying the rates of error-free and mutational COs in thousands of cells, enabling us to explore enrichment patterns of genomic and epigenomic features. The throughput of sci-L3-Strand-seq also gave us the ability to measure subtle phenotypes, opening the door for future large mutational screens. Furthermore, mapping clonal lineages provided insights into the temporal order of certain genome instability events, showcasing the potential to dissect cancer evolution. Altogether, we show the wide applicability of sci-L3-Strand-seq to the study of DNA repair and structural variations.

HNRNPU is an RNA-binding protein with diverse roles in transcriptional and post-transcriptional regulation. Pathogenic genetic variants of HNRNPU cause a severe neurodevelopmental disorder (NDD), but the underlying molecular mechanisms are unclear. Here, we comprehensively investigate the HNRNPU molecular interactome by integrating protein-protein interaction (PPI) mapping, RNA target identification, and genome-wide DNA methylation profiling in human neuroepithelial stem cells and differentiating neural cells. We identified extensive HNRNPU-centered networks, including an association with the mammalian SWI/SNF chromatin-remodeling complex, and uncovered a previously unrecognized role in translation. We present evidence that HNRNPU associates with messenger RNAs (mRNAs) encoding proteins important for neuronal development, including several linked to NDDs. Silencing HNRNPU reprogrammed methylation dynamics at regulatory regions, particularly at active and bivalent promoters of neurodevelopmental transcription factors. Integrative analysis across PPI, RNA, and methylome datasets identified 19 converging genes at all three molecular levels, including NDD genes within the SWI/SNF complex, SMARCA4 and SMARCC2, and RNA-processing machinery such as SYNCRIP. Together, these data showcase HNRNPU as a central coordinator of RNA metabolism and epigenetic remodeling during neural differentiation, linking RNA-binding, chromatin organization, and DNA methylation to the pathogenesis of HNRNPU-related NDDs.