Nucleic Acids Research最新文献

The characterization of protein-DNA interactions underpinning fundamental biological processes requires methods that are laborious and time-consuming. Here, we introduce a rapid and instrument-free assay leveraging on GFP-tagged proteins and the lateral flow assay principle to examine protein-DNA interactions. The rapid protein-DNA interaction test (R-PNAI-T) detects complexes using a dipstick in ∼15 min. Validation of the R-PNAI-T with bacterial proteins involved in replication and transcription demonstrated its applicability for diverse protein-DNA interactions, achieving a remarkable detection sensitivity of ∼0.7 fmol with the Escherichia coli Tus protein. Analysis of these protein interactions with their specific target DNA sequences highlighted the capability of the R-PNAI-T to discern subtle differences in affinity, providing valuable comparative data. The R-PNAI-T is robust, retaining functionality in human serum and bacterial lysates. The assay showed tolerance towards biotin contaminants, expanding its use for quality control in protein purification processes and tracking complexes in biological samples. To demonstrate versatility, we applied the R-PNAI-T with polymerase chain reaction-amplified DNA to probe the putative origin of replication in Burkholderia pseudomallei, confirming a functional interaction between the DnaA initiator protein and the DnaA box in this bacterium. Overall, the R-PNAI-T is versatile, cost-effective, and user-friendly, offering broad applications in biological research and biotechnology.

CRISPR-Cas13 systems, harnessed for RNA-guided transcriptome editing, hold significant promise for clinical and in vivo therapeutic applications. However, understanding their in vivo target specificity and recognition rules remains a challenge. In this study, we employed the uSpyCLIP method, which enhances sensitivity and specificity for identifying RNA-binding protein (RBP) binding sites, to map the transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) in HEK293T cells, using a variety of single guide RNAs (gRNAs). Surprisingly, we identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes. These gRNA-independent off-target sites exhibited distinct RNA structural and sequence signatures: dPspCas13b's gRNA-independent binding was associated with specific RNA structural features, while dRfxCas13d's was linked to unique sequence motifs. Analysis of gRNA-dependent off-target sites revealed the crucial role of the DR-distal and middle regions of the gRNA in determining binding specificity. Further analysis demonstrated that some off-target binding events led to changes in gene expression at the messenger RNA and/or protein level. Collectively, our findings provide important insights into the characteristics of gRNA-dependent and gRNA-independent off-target binding for PspCas13b and RfxCas13d, offering valuable guidance for optimizing Cas13 and gRNA design in future applications.

The process of male germ cell development is a central determinant of spermatogenesis. Nevertheless, the genetic regulatory mechanisms underlying male germ cell development in mammals remain largely unclear. In this study, employing a germ cell-specific Hnrnpk knockout mouse model combined with multi-omics analyses, we identified hnRNPK as a key factor necessary for maintaining normal development in differentiating spermatogonia. Phenotypically, adult mice with germ cell-specific hnRNPK deletion exhibited infertility, characterized by a near-complete absence of spermatocytes in the seminiferous tubules. Single-cell RNA sequencing (scRNA-seq) analysis revealed that hnRNPK deletion induced cell-cycle dysregulation in differentiating spermatogonia, triggering apoptotic cell death. As a consequence, the population of differentiating spermatogonia in the testes is markedly diminished, and these cells fail to undergo proper maturation or successfully enter meiosis. Mechanistically, cytoplasmic hnRNPK exerts its regulatory function at the post-transcriptional level, regulating the translation efficiency (TE) of genes involved in meiosis, the cell cycle, and transcriptional regulation. Furthermore, hnRNPK interacts with and colocalizes with DAZL at the 40S ribosome, thereby modulating the initiation of target messenger RNA translation. In the nucleus, hnRNPK interacts with splicing factors and participates in the splicing of target genes related to germ cell differentiation and meiosis. Collectively, these findings emphasize the functional role and mechanistic involvement of hnRNPK in differentiating spermatogonia, providing valuable insights into the post-transcriptional regulatory mechanisms that govern male germ cell development.

Nucleotide excision repair (NER) is the key pathway for the removal of DNA damage induced by UV irradiation and chemotherapeutic reagents. Protein-protein interactions are crucial for the dynamic and coordinated assembly of the proteins involved in DNA lesions. Here we focus on the role of interactions between the multi-subunit helicase/translocase complex TFIIH and the 3' endonuclease XPG. We show that XPG interacts with the p62 and XPD subunits of TFIIH through its long spacer region bridging its split active site. We show that interactions between three acidic regions of XPG and the Pleckstrin homology (PH) domain of p62 are of moderate importance for NER, while defects in the interactions with XPD fail to pull-down TFIIH and strongly reduce NER activity. These p62 and XPD interface mutations additively reduce NER activity. Unexpectedly, we show that these interactions did not impair the recruitment of XPG but instead were defective in the formation of a catalytically competent NER complex and in triggering the incision 5' to the lesion by ERCC1-XPF. Our studies provide fundamental insights into how interactions between TFIIH and XPG contribute to the NER pathway and, more generally, how modular protein-protein interactions control each step along the NER reaction coordinate.

Certain DNA bacteriophages exhibit a complete substitution of their genomic adenine (A) by 2-aminoadenine (Z), forming three hydrogen bonds with thymine. dZTP biosynthesis is performed by a phage-encoded 2-amino adenylosuccinate synthetase (PurZ) whereas a Z-specific DNA polymerase I (DpoZ) has been shown to incorporate the dZTP. Our investigations into the nucleotide metabolism of Z-bacteriophages, integrating modeling, biochemical, and phylogenetic approaches, reveal novel enzymatic activities. We characterized two distinct enzymes that both hydrolyze dATP and dGTP, and a DmtZ enzyme with dual activity. DmtZ acts as a dAMP-specific hydrolase, converting dAMP to adenine, and uniquely transfers deoxyribose 5-phosphate from dAMP to the Z base to produce dZMP, which is subsequently converted to dZTP. This dual functionality marks DmtZ as the first enzyme in the nucleoside deoxyribosyltransferase (NDT) family with such a mechanism and uncovers a novel biosynthetic route for dZTP. Phylogenetic analyses indicate multiple independent acquisitions of enzymes involved in nucleotide metabolism, occurring after PurZ acquisition, yet converging on equivalent metabolic functions. Deciphering these propagation mechanisms in DNA-modified bacteriophages illuminates functional diversity in viral metabolism and a striking example of functional convergence.

Activation of the ATR-dependent DNA damage response (ATR-DDR) is well characterized; however, the molecular mechanisms underlying its maintenance and inactivation remain largely elusive. Rhino is the least understood component of ATR-DDR. Structural modeling and binding free energy calculations revealed structural remodeling involving Rad17, Rad9-Hus1-Rad1 (9-1-1), and Rhino during ATR-DDR progression. Biochemical and computational analyses revealed the competitive binding of Rad17 and Rhino to the 9-1-1 complex, suggesting a structural transition from the Rad17-9-1-1 complex to the Rhino-9-1-1 complex. The presence of two conserved KYxxL+ motifs in Rhino suggests that it bridges the two 9-1-1 complexes. This enables the polymerization of multiple 9-1-1 complexes through Rhino and explains the long-standing discrepancy between the conventional model and experimental observations of Rad17 and Rad9 foci. Furthermore, structural analysis of the Rad9 C-terminal tail revealed its ability to compete with both Rhino and Rad17, leading to disassembly of the checkpoint complex and providing a mechanism for checkpoint inactivation. Quantum chemical calculations revealed comparable binding free energies for intermediate complexes. These observations suggest that the Rad17-9-1-1-Rhino complex undergoes energetically equivalent structural transitions, providing a mechanistic basis for the sequential progression of ATR-DDR.

Transposable elements (TEs) drive genomic innovation, but their dynamics in non-model species remain unclear. Here, we integrated multi-omics data to explore TE dynamics in Drosophila virilis, an important model for repetitive DNA research. By combining computational predictions with manual curation, we identified 100 TE families and delineated three temporal waves of TE mobilization: recent activity, speciation-associated, and ancient invasions. TEs in D. virilis dynamically colonise both euchromatin and heterochromatin, suggesting heterochromatin is not solely a repository for degenerate repeats. While most TEs are widespread across strains, some exhibit strain-specific expansions, indicating varied activity and silencing. We found substantial evidence for horizontal transfer of TEs among close relatives, demonstrating that the D. virilis species group functions effectively as a TE "ecosystem", allowing for recurrent invasion, loss, and re-invasion of TE lineages across the group. Epigenetic profiling revealed that H3K9me3 spreading from TEs represses adjacent genes in a distance-dependent manner, influenced by insertion length and genomic context, affecting developmental and metabolic genes. We also discovered the first spontaneous polymorphic inversion in D. virilis linked to retrotransposons. Our findings illuminate TEs as drivers of genomic innovation, influencing gene regulation and evolutionary trajectories, providing a framework for studying TE dynamics across animal species.

We present a simple model for analyzing and interpreting data from kinetic experiments that measure engaged RNA polymerase occupancy. The framework represents the densities of nascent transcripts within the pause region and the gene body as steady-state values determined by four key transcriptional processes: initiation, pause release, premature termination, and elongation. We validate the model's predictions using data from experiments that rapidly inhibit initiation and pause release. The model successfully classified factors based on the steps in early transcription that they regulate, confirming TBP and ZNF143 as initiation factors and heat shock factor and glucocorticoid receptor as pause release factors. We found that most paused polymerases terminate and paused polymerases are short-lived with half lives less than a minute. We make this model available as software to serve as a quantitative tool for determining the kinetic mechanisms of transcriptional regulation.

Misassigned Mg2+ ions are pervasive in RNA structural databases, obscuring mechanistic interpretation, undermining comparative analyses, and compromising machine-learning training sets. Here, we present Cat_Wiz, a Coot-integrated, stereochemistry-guided toolkit that facilitates the localization, diagnosis, correction, and annotation of Mg2+ binding sites. Cat_Wiz comprises three modules: MG_diagnosis,which validates and regularizes existing assignments; MG_detect, which identifies unmodeled ion binding sites; and MG_clamp, which classifies recurrent Mg2+ clamp motifs. Cat_Wiz also includes a complete binding site annotation system. The stereochemical principles implemented in Cat_Wiz were derived from an earlier analysis of the 1.55 Å resolution Escherichia coli ribosome and from surveys of the Cambridge Structural Database (CSD). These principles provide a robust experimental foundation for characterizing Mg2+ binding sites. Applications to ribosomes, hammerhead ribozymes, group I introns, and quaternary RNA assemblies demonstrate that Cat_Wiz rapidly locates overlooked ions, corrects misassignments, and improves stereochemical fidelity in hours rather than days. Beyond refinement, Cat_Wiz generates curated data that can seed diverse machine learning and artificial intelligence (AI) models. This transparent, cost-effective framework establishes reproducible standards for RNA-ion assignments and will drive progress in the design of RNA 3D architectures through the identification of unique Mg2+-dependent backbone folds. Cat_Wiz, that is based on universal stereochemical principles, applies also to Mg2+ binding sites in proteins and related biomolecular systems.