Nucleic Acids Research

BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.

Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the types and lengths of DNA ends following cleavage. Here, we investigated Cas12a's ability to produce different ends and if they can be used to redistribute editing outcomes. We determined the sites and rates of target cleavage by Cas12a in vitro by combining kinetic modeling with nucleotide-resolution assays. For the first time, we show that trimming - repeated cleavage of an already cut target - occurs about 4x faster than initial cleavage; it also presents alternative DNA end structures for cellular repair. We next introduced specific mismatches to the gRNA. Cas12a maintained fast target cleavage, but changed where the target was cleaved and how quickly it was trimmed, compared to matched gRNA. We exploited the differences in cleavage dynamics between matched and mismatched gRNAs to develop reprogrammed gRNAs, i.e. rpgRNAs. Intentionally-mismatched rpgRNAs retained the high editing efficiency observed with traditional gRNAs. However, they redirected editing between in-frame and out-of-frame outcomes to enhance gene knockout success across genes. Reprogrammed gRNAs offer an efficient way to steer editing toward such preferred outcomes, while retaining the simplicity of gene editing with CRISPR-Cas nucleases.

Canonical McrBC enzymes are nucleotide-powered, motor-driven endonucleases that bind and cleave modified bacteriophage DNA. Non-canonical McrBC homologs like LlaI and BsuMI are distinguished by a unique three-gene organization and the ability to target DNA site-specifically. Here, we report the atomic-resolution crystal structures of the DNA-binding module LlaI.R1 and AAA+ motor LlaI.R2 from the Lactococcus lactis LlaI anti-phage defense system. The crystallized LlaI.R2 hexamer traps two distinct active site conformations that correlate to different states of the nucleotide hydrolysis cycle and reveal that the organization of the critical catalytic machinery present in canonical McrB homologs is also conserved in non-canonical R2 proteins. Although canonical McrB homologs are strictly GTP-specific, we find that the R2 proteins from LlaI and BsuMI do not discriminate between different nucleotides, even when in complex with their respective R1 partners. Using mutagenesis, we define surfaces on the LlaI.R1 structure that are critical for DNA-binding and interaction with LlaI.R2. These observations support computational modelling of the assembled LlaI restriction system bound to DNA. Together, our data provide new insights into the evolution of nucleotide specificity in McrBC restriction complexes and the molecular mechanisms governing McrBC-catalyzed DNA translocation and cleavage.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

mRNAs can form stable structures that need to be resolved to facilitate translation. During translation initiation in mammals, the scanning 48S complex requires the helicase activity of DHX29 to unwind stable mRNA structures that cannot be resolved by eIF4A. Here, we show that the yeast DHX29 homolog, Ylr419w (Dhx29), has a similar function during translation on elongating 80S ribosomes. Cryo-EM analyses show that the Dhx29 helicase module is positioned at the mRNA entry channel to engage mRNA, while its double-stranded RNA-binding domain (dsRBD) senses hairpin-forming mRNA in the ribosomal A-site. By selective ribosome profiling, we observed that Dhx29 is associated with transcripts that form RNA structures, such as stable tetraloops. Dhx29 mutants with perturbed helicase activity enrich 80S with hairpins in the A-site, as well as ribosome collisions, while a mutant lacking the N-terminal dsRBD sensor domain loses the specificity for such ribosomes. We thus propose that Dhx29 functions in translation elongation by resolving structured mRNA formed in the ribosomal A-site through its 3'-5' helicase activity and pulling on the mRNA from its 3' end.

Replication-associated errors can cause DNA damage to accumulate on the newly synthesized strand over time. In specific cases such as stem cells, retention of the immortal strand used as template preserves one daughter cell into pluripotency while correlating with terminal differentiation of the damage one. In somatic cells, DNA damage distribution after mitosis remains unclear. Here, we uncovered a mechanism of non-random segregation of the DNA damage marker gH2AX occurring during a single cell division cycle. Replication stress using hydroxyurea (HU) upon release into S phase in RPE-1, BJ, hCEC D29 and fibroblasts showed reproducible Non-Random Segregation (NRS) of gH2AX in the ensuing G1, a phenotype not observed in any of the cancer cell lines analyzed. Notably, removal of R-loops led to a reduction of cells with NRS, whether RNaseH1 was over-expressed globally or exclusively targeted to centromeres, indicating that centromeric DNA-RNA hybrids contribute to NRS of the damage. In line with our previous evidence of centromeric chromatin disruption leading to R-loops, rapid removal of the histone H3 variant CENP-A causes damage and NRS, although to a lower extent than HU alone. This implies that additional mechanisms contribute to centromeric R-loops and NRS of damage in the daughter cells upon mitotic exit. Mechanistically, chemical inhibition of the catalytic activity of Rad51 led to a significant drop in NRS without a change in the total amount of damaged cells, implying involvement of the Homologous Recombination (HR) pathway to accumulation of gH2AX to only one chromatid. In turn, this affects the spindle-kinetochore with a measurable length asymmetry, inducing mechanical and/or epigenetic signals that affect the orientation of the sister chromatids on the metaphase plate to bias segregation. Altogether, we found replication-induced asymmetric segregation of DNA damage during mitosis that is influenced by centromeric R-loops, Rad51 activity and spindle dynamics, with implications on cell fate, chromosome and genome stability in the daughter cells.

DNMT3A is a de novo DNA methyltransferase whose recruitment to chromatin regulates its function. Missense mutations within the chromatin-binding PWWP domain are associated with diverse human disorders, yet how mutations in the same domain produce distinct phenotypes remains unclear. Here we systematically characterise 19 clinically reported mutations in the PWWP domain of DNMT3A that are associated with Heyn-Sproul-Jackson syndrome (HESJAS), paraganglioma (PG) and clonal haematopoiesis (CH). We show that all PWWP-domain mutations associated with HESJAS abolished interaction with H3K36me2 modified nucleosomes, defining this as a consistent biochemical feature of HESJAS. In contrast, mutations from all disease classes differentially altered DNA binding of the PWWP domain, driven by alterations in the net charge of the domain. However, these effects are largely overcome by inclusion of the DNNMT3A1 N-terminal region, which is absent from its embryonic isoform, suggesting that PWWP mutations may differentially affect DNMT3A function through development. Changes in the thermal stability of the isolated PWWP domain mutants did not directly translate into altered stability of full-length DNMT3A1 in cells. We show that HESJAS mutations can affect the intramolecular interaction between the PWWP and adjacent ADD domain, an interaction proposed to contribute to the autoinhibitory function of the ADD domain. However, not all mutations behaved in the same way, suggesting that multiple factors govern the intramolecular autoinhibition of DNMT3A. Together, this study advances our understanding of the molecular mechanisms by which DNMT3A PWWP-domain mutations are mechanistically heterogeneous, providing a biochemical framework that contributes to distinct disease phenotypes.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Pre-mRNA splicing is essential for gene expression, yet how disruption of core spliceosomal factors produces tissue- and developmental stage-specific phenotypes remains poorly understood. Here, we investigated the in vivo function of the conserved spliceosomal kinase PRPF-4 in C. elegans using endogenous reporter analysis, conditional protein depletion, and transcriptome-wide analysis of alternative splicing and gene expression. We found that PRPF-4 is broadly expressed throughout development and is continuously required for postembryonic development, with distinct requirements in the pharynx, nervous system, and germline. Acute PRPF-4 depletion rapidly disrupts alternative splicing across thousands of transcripts, with exon skipping representing the predominant class of affected events. In addition, PRPF-4 depletion results in a robust transcriptome shift with induction of components of the spliceosome and repression of ciliary and ion transport-related transcripts. These findings establish PRPF-4 as a central regulator of RNA metabolism and demonstrate the far-reaching effects on gene expression caused by loss of core spliceosomal components.

Genome-wide coexpression analysis of intact tissue samples is a powerful approach for identifying reproducible signatures of cell types and states, since it can survey vast numbers of individuals, cells, and transcripts. However, it can be difficult to optimize gene coexpression network construction and compare results from independent analyses. To address these challenges, we developed OMICON (theomicon.ucsf.edu) for research on human brain gene coexpression networks. OMICON contains gene expression data from >17K normal and neoplastic human brain samples with standardized metadata. Systematic analysis of independent datasets identified >250K gene coexpression modules, which were characterized and compared via enrichment analysis with >40K gene sets. All modules are discoverable via an advanced search engine that can filter by genes, metadata, and enrichment results. Analyses can also be browsed with an interactive workflow visualization tool, and users can communicate within OMICON using @mention functionality to support communal research on human brain gene coexpression networks.

The interpretation of missense variants remains a major challenge in clinical genetics. "Meta-domains" aggregate population and pathogenic variation across homologous Pfam domain instances in the human proteome, providing per-residue context for interpreting variants of uncertain significance (VUS). Our 2019 implementation, MetaDome, is widely used and named in clinical variant-classification guidelines. Here we present the MetaDome 2027 update, featuring a comprehensively updated dataset and GRCh38 support. The redesigned pipeline enables incremental updates of GENCODE, UniProtKB/Swiss-Prot, Pfam, gnomAD, and ClinVar while maintaining 100% sequence-identity gene-to-protein mapping. Annotated Pfam domain instances grew 14.9% from 71,419 to 82,069 and meta-domain-eligible Pfam families ([≥]2 human occurrences) by 73.3% from 3,334 to 5,778; Pfam domains are annotated to 92% of human proteins. Approximately 43% of mapped protein-coding nucleotides (14.3 million in GRCh38, 13.8 million in GRCh37) are in a meta-domain; in GRCh38 67.9% (37,692 of 55,548) of pathogenic or likely pathogenic ClinVar missense variants fall at such a position. We show how MetaDome helped reclassify a de novo missense VUS in RALA and identify 52,463 ClinVar missense VUS for which meta-domains supply otherwise unavailable pathogenic evidence. MetaDome is freely available at www.metadome.app.

Transcription factor Ets2 coalesce with the NF-{kappa}B pathway to regulate gene expression in specific signaling contexts. IKK2/{beta}-mediated phosphorylation events critically regulate the NF-{kappa}B pathway. However, any link between Ets2 and IKK2 remains elusive. Here we report Ets2 as a direct substrate of IKK2. In-vitro kinase assays using deletion constructs, high resolution MS-MS and site directed mutagenesis identified S295 as a prominent phosphorylation site distal to the DNA binding domain, substitution of which to phosphor-mimetic Glutamate triggers further phosphorylation of Ets2. MD simulations clearly indicate conformational constriction of the otherwise disordered N-terminal region and inhibition of DNA binding activity upon phosphorylation, which was further confirmed by Electrophoretic mobility shift assays. Our results uncover a phosphoregulatory connection between Ets2 and IKK2.

Biological aging is accompanied by systematic changes in epigenetic modifications and chromatin organization. The reversal of the effects of aging, rejuvenation, is experimentally achieved by the transient induction of factors that modify these marks in cells and organisms. Here, we show that key features of rejuvenation experiments emerge from the biophysical interplay between dynamic epigenetic marks and the three-dimensional conformation of chromatin. Using a minimal field theory and molecular dynamics simulations, we show that the system responds in three distinct temporal regimes. The intermediary regime fulfills necessary conditions for successful rejuvenation. In this regime, the system spends time near a separatrix, allowing for high epigenetic plasticity, while memory retained in the chromatin conformation enables restoration of the original epigenetic correlations. Analysis of sequencing data further supports the predicted coupling between chromatin compaction and epigenetic correlations. Our results provide a physical explanation for how rejuvenation may remodel age-associated epigenetic states without irreversibly erasing cellular identity. We identify a general mechanism by which memory stored in a slow structural variable permits reversible remodeling of a faster internal state.

Phage tail-like elements (PTEs) -- tailocins, bacterial type VI secretion systems (T6SS), and extracellular contractile injection systems (eCIS) -- are contractile nanomachines that bacteria use to kill their neighbors and compete within their micro-ecosystems. PTEs help shape microbial community composition. Most PTE detection tools only detect a single PTE class. Moreover, most tailocin detection methods are largely restricted to Pseudomonas, leaving a key part of tailocin diversity uncharacterized. In this work, we present PhageTAILor (https://github.com/hjcho-bio/PhageTAILor), an integrative and fully automated pipeline that detects and classifies prophages and 3 PTE classes from bacterial genomes. PhageTAILor combines a 6-detector homology-based candidate search (geNomad, tail-gene, PHROGs-tail, SecReT6, eCIStem, and a divergence-tolerant tail-HMM detector) with a LightGBM classifier comprising 1 multiclass and 3 binary heads, trained on 6,501 bacterial genomes carrying 13,082 prophages and PTEs. A phylogeny-free feature matrix used in our model keeps predictions reproducible between model construction and user inference. PhageTAILor performs strongly at the genome level and generalizes beyond its Pseudomonas-rich training set. On a 76-strain cross-clade benchmark, PhageTAILor detected tailocins at F1 = 0.955. Furthermore, it identified 12 of 13 experimentally validated tailocins spanning five genera versus 2 of 13 for a Pseudomonas-restricted tool TattleTail. PhageTAILor also demonstrated sensitivity equivalent to viral detection tool geNomad while avoiding its higher false-positive rate. Applied to 7,925 plant- and soil-associated bacterial isolates, PhageTAILor showed that prophages in the phyllosphere and tailocins in plant-associated bacteria, whereas eCIS are enriched in soil. PhageTAILor is distributed as an open-source, modular pipeline with a command-line interface.

Plant-specific RNA Polymerase V (Pol V) transcribes noncoding RNAs in the RNA-directed DNA methylation pathway, thereby influencing gene expression and genome stability by controlling de novo DNA methylation. However, the mechanisms governing precise chromatin localization and transcriptional activities of Pol V remain elusive. Here we show that Pol V localization is spatially constrained by the chromatin regulators microrchidia (MORC) and MORPHEUS' MOLECULE 1 (MOM1). MORC and MOM1 promote Pol V occupancy at sites near active chromatin, whereas their loss leads to redistribution of Pol V into CMT3-enriched heterochromatin, accompanied by noncoding RNA transcription, small RNA production and DNA methylation. Our findings reveal a combinatorial model in which recruitment, spatial constraint and DNA methylation feedback collectively define Pol V chromatin distribution and epigenetic function.

Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.

Scientific agents can produce plausible answers while remaining unable to establish whether the computation behind an answer is executable, recoverable, or reproducible. We present BloClaw, an AI4S workstation built around a simple principle: a scientific agent should know what it can do, show how it did it, and state what remains unvalidated. Each capability declares an execution state, input constraints, dependencies, expected outputs, and scientific limitations. Natural-language requests are translated into structured tasks, validated against this registry, executed through scientific tools, and recorded in a provenance-aware Living Lab Notebook. The system is designed to detect invalid inputs, failed tool calls, missing dependencies, and remote timeouts, and to route them to repair, retry, or escalation. The implemented and tested scope comprises RDKit-based molecular property and rule screening, protein structure analysis, docking-pose inspection, 3D visualization, and structured reporting. We demonstrate the workflow on a PubChem-retrieved osimertinib structure and a supplied 6LU7 docking artifact: the former yields deterministic descriptors (molecular weight 499.619 Da, cLogP 4.5098, TPSA 87.55 A^2), while the latter contains 2,387 protein ATOM records, 309 residues, and nine pose records. These examples are workflow demonstrations, not efficacy or affinity studies. Beyond retrospective prediction, the manuscript specifies a prior-minimized constructive mode in which a desired function is compiled into explicit physical, chemical, and systems constraints, candidate mechanisms are simulated, and observations are reintroduced for calibration and falsification; this is a proposed extension rather than a result of the present case studies. We describe an evaluation protocol that compares BloClaw with a standard single-agent workflow and fixed-script execution using task completion, scientific correctness, recovery success, provenance completeness, reproducibility, human review time, latency, and cost. This manuscript reports the system design, verified capability boundary, deterministic software artifacts, and a reproducible evaluation protocol; it does not claim benchmark improvements before those experiments are run. BloClaw is an execution and accountability layer for AI-assisted research, complementing expert review and experimental validation rather than replacing them.

Plasmodium falciparum heat shock protein 90 (PfHsp90) is a promising antimalarial target, but the molecular pathways influenced by its inhibition remain poorly understood. Herein, we leveraged chemoproteomic profiling employing geldanamycin and XL888 Hsp90 inhibitors to investigate proteins and pathways dependent on the chaperone during the Plasmodium blood stage. This study revealed 131 proteins reduced in abundance after inhibition, of which 40% co-immunoprecipitated with PfHsp90. Bioinformatic analyses identified DNA replication as the most enriched pathway. This link was investigated in phenotypic studies demonstrating reduced parasite DNA content after PfHsp90 inhibition. To assess nascent DNA synthesis, we utilized a 7-deaza-7-ethynyl-2'-deoxyadenosine (EdA) assay, yielding dual-stage attenuation of nucleoside incorporation following Hsp90 inhibition. We further show that parasite co-treatment with Hsp90 and DNA replication inhibitors produces synergistic interactions, highlighting the therapeutic potential of the discovered link. Overall, these findings expand our understanding of PfHsp90 function and uncover novel PfHsp90-dependent pathways.

In linear polysomes, excluded-volume interactions among ribosomes can induce dimensional reduction of mRNA. Yet linear architectures allow steric stress to relax at open ends-- limiting how strongly crowding can remodel the mRNA's structure and dynamics. Using coarse-grained molecular-dynamics simulations, we compare circular and linear polysomes over a range of ribosome densities. Circular closure selects a predominantly quasi-planar global conformational ensemble, as indicated by a shape dimensionality dshape {approx} 2 over a range of ribosome densities. Crucially, circular topology and ribosome crowding act cooperatively to suppress structural fluctuations. While closure alone or linear crowding reduces relative global size fluctuations ({Delta}Rg/Rg) only to {approx} 0.16, their combined effect drives this fluctuation down to {approx} 0.07. Within this stabilized architecture, increasing ribosome density drives a distinct in-plane reorganization: the ring becomes more isotropic, global size fluctuations are strongly suppressed, and the scaling exponent increases toward {nu} [~=] 0.74 - 0.77, consistent with two-dimensional self-avoiding walk-like value over the accessible finite-size window, 1000 [≤] N [≤] 4969. Closure shortens the radius-of-gyration decorrelation time of circular polysomes by 40-fold relative to matched linear systems, reflecting the topological elimination of free ends. Within this closureselected ensemble, ribosome crowding further reduces the decorrelation time by up to 20% at the highest density. A fluctuation-informed crossover model links the density dependence of the global scaling exponent to inter-ribosomal subchain statistics. These results distinguish the geometric role of circular closure from the density-dependent steric response that it enables, revealing a confined yet dynamically responsive conformational regime for circular polysomes.