Nucleic Acids Research

5' nicotinamide adenine dinucleotide (NAD+) caps are one of the most common metabolites derived non-canonical caps reported on bacterial RNAs. Multiple decapping proteins are known to regulate the stability of NAD+ capped transcripts. However, no other proteins have been identified that preferentially interact with these NAD+ caps, and mechanistic details of the cap-dependent recognition remain poorly understood. Using an affinity capture approach, we identified multiple E. coli proteins that selectively recognize NAD+ caps, including the ATP-dependent RNA helicase, CsdA. CsdA preferentially interacts directly with NAD+ capped RNAs and can discriminate between 5' NAD+ capped and 5' triphosphate end transcripts. Binding to NAD+ capped RNA versus 5' triphosphate RNA more greatly enhances the ATPase activity of CsdA and the presence of NAD+ caps on transcripts modulates the ability of CsdA to form RNA condensates. Furthermore, we find that CsdA enhances the decapping activity of the NADH hydrolase NudC, suggesting CsdA plays a role in regulating the degradation of NAD+ capped transcripts. CsdA is the first identified NAD+ cap reader protein and its preference for binding NAD+ capped RNA provides a mechanism by which E. coli cells link RNA stability to the identity of the 5' cap. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/730015v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@115a057org.highwire.dtl.DTLVardef@d1c2c6org.highwire.dtl.DTLVardef@14a6d2dorg.highwire.dtl.DTLVardef@145ccff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Identifying DNA replication origins in human and other metazoan genomes has been challenging, as highlighted by the fact that various methods for mapping them have produced conflicting results. A popular method, short nascent strand sequencing (SNS-seq), enriches newly replicated short single-stranded DNA by size selection and{lambda} -exonuclease ({lambda}-exo) digestion of parental DNA. Surprisingly, SNS-seq has never been validated in Saccharomyces cerevisiae where origins have been well characterized genome-wide. We improved the SNS-seq protocol through biochemical optimization and benchmarked its origin-mapping sensitivity and precision with traditional SNS-seq in asynchronous populations of S. cerevisiae. The improved SNS-seq protocol significantly enhanced the enrichment of origin-derived DNA. Strikingly, the traditional SNS-seq failed to detect known origins and instead enriched non-origin DNA, likely arising from RNA:DNA hybrids. These findings have important implications for the interpretation of previously published datasets that rely on{lambda} -exo for origin mapping. Overall, our biochemical and genomic analyses help unravel the mystery of the inconsistencies between SNS-seq and other techniques used to map DNA replication origins genome-wide.

Escherichia coli couples the initiation of DNA replication with cell size by modulating the activity of the replication initiator protein DnaA. The activity of DnaA is regulated by both its interconversion between an active and inactive form and its titration on binding sites on the chromosome. Whereas its interconversion has been thoroughly studied, the extent to which DnaA titration can control replication initiation is poorly understood. Here, we describe the control of E. coli DNA replication via titration by modulating the expression of an always-active DnaA variant in four growth conditions. While we obtained stable cell cycles during slow growth, faster growth associated with overlapping replication forks led to replicative instability and DNA damage. Overall, our results provide insights into the limits of titration-based systems in the control of genome replication and their potential role in the evolutionary trajectory of E. coli. Finally, this study provides design principles for a simplified, titration-only regulatory mechanism for DNA replication in synthetic cells.

Replication risk sequences (RRS) are recently discovered genomic structural elements that trigger post-replication gap formation during replisome passage. In E. coli, the two RRS elements are nearly perfect 222-bp G-quadruplex-containing repeats that flank the terminal domain and are highly conserved in both sequence and genomic position across enterobacteria. We report here the first direct visualisation of RRS function in vitro using single-molecule methods. When the G4 strand of the RRS element is positioned on the lagging-strand template, gaps are formed essentially every time a replisome encounters it. An increase in ssDNA in the synthesised DNA is readily seen using ssGAP-seq methods. When the G-quadruplex strand of the RRS is positioned on the leading-strand template, gaps are formed, albeit at lower frequency. However, the continued DNA synthesis in a rolling-circle assay indicates that the gaps are still formed on the lagging strand, indicating that the RRS complementary strand has a significant but reduced capacity to form a structure that triggers lesion skipping. The results document the potency of the RRS as a trigger for gap formation, suggesting a possible function for at least some eukaryotic G-quadruplexes.

The eukaryotic ribosomal genes are multi-copy genes, transcribed from the rDNA, and approximately one third of them is actively transcribed in differentiated cells. A number of lncRNAs have been identified from the intergenic spacer between the rRNA genes, among those the spacer RNA and PAPAS that are involved silencing of rRNA gene copies by altering the chromatin configuration. Here, we have identified lncRNAs that are transcribed from the human rDNA loci and modulate the loci; IGS38 positively regulates rRNA gene transcription by associating to the 47S rRNA gene promoter and modulating the rRNA promoter accessibility while IGS32as associates with heterochromatin. IGS38 binds to the 47S gene promoter through the RNA pol I factors TAF1C and RRN3 as well as the Williams Syndrome Transcription Factor (WSTF), a component of the B-WICH chromatin remodelling complex. The increased accessibility of the promoter stabilises the architectural protein Upstream Binding Factor (UBF) at the rRNA promoter, thereby facilitating RNA pol I promoter escape. Furthermore, IGS38 knock down displays and increased dsRNA abundance in the cytoplasm with a weak induction of the dsRNA sensor OAS2, typically induced by interferon and viral dsRNA. Overall, the both IGS38 and IGS32as are chromatin associated lncRNAs involved in rDNA chromatin changes, and IGS38 is stimulating, together with WSTF, rRNA gene transcription in human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/722362v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d4159org.highwire.dtl.DTLVardef@fd773forg.highwire.dtl.DTLVardef@a0030dorg.highwire.dtl.DTLVardef@1285301_HPS_FORMAT_FIGEXP M_FIG C_FIG IGS stabilises 47S rRNA transcription, disruption of IGS38 expression leads to the release of dsRNA in the cytoplasm and a weak immune activation of OAS2. Created by biorender (https://biorender.com/shortURL)

Ribozyme-based permuted intron-exon (PIE) systems offer a protein-independent route to circRNA production, but existing platforms require elevated temperatures that promote RNA degradation. Here we report the first application of the Candida albicans mitochondrial large subunit (C.a.mtLSU) group I intron as a PIE platform for circRNA synthesis, which we term PCanPIE (Pyle lab Candida PIE). We evaluated three peripheral stems, P5, P6b, and P8, as permutation sites and demonstrated that all three support circularization under near-physiological conditions (25{degrees}C, 6 mM MgCl2), without the 55{degrees}C heating step required by existing PIE systems. Kinetic analysis revealed that permutation site does not affect the observed splicing rate constant but does influence PCanPIE folding and therefore influences circularization efficiency. The P6b permutation yielded the highest circularization efficiency, with 95 % of the precursor splicing to produce circRNA. Optimization of spacer sequences flanking the circRNA payload eliminated interference from structured native exon sequences and enabled efficient circularization of RNAs up to 1,657 nt, including structured, repetitive, and naturally occurring sequences. Together, these results establish PCanPIE as a versatile and near-physiologically active addition to the group I intron PIE toolkit.

Deinococcus radiodurans harbors a largely classical bacterial DNA repair machinery yet displays exceptional resistance to ultra-violet and ionizing radiation. To investigate whether crosstalk between its DNA repair pathways contributes to this phenotype, we mapped putative interactions between the nucleotide excision repair (NER) and base excision repair (BER) pathways, which together are responsible for the removal of nucleobase lesions. Using a bacterial two-hybrid system, we identified multiple direct interactions between NER and BER proteins, notably involving the two UvrA variants, and validated these interactions in vitro. Furthermore, functional analyses revealed that NER interferes with the BER-mediated removal of oxidized guanines by the Fpg DNA glycosylase, likely through competition for DNA binding and sequestration of Fpg. Finally, UvrB and UvrC were found to further process the Fpg incision product in an ATP-dependent, UvrA1-independent manner. Together, these results demonstrate a multi-level crosstalk between NER and BER in D. radiodurans, which may contribute to its extraordinary DNA repair capacity. To our knowledge, this represents the first evidence of such a complex interplay in bacteria.

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this "OTTR-CLASH" method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/738487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13bc276org.highwire.dtl.DTLVardef@5beb41org.highwire.dtl.DTLVardef@b204e5org.highwire.dtl.DTLVardef@15f747d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Defining high-confidence RNA interaction sites for specific proteins is essential to understand RNA biology, but existing methods face trade-offs between specificity, sensitivity, and experimental accessibility. Here, we present fluorescent Cross-linking and analysis of cDNAs (fCRAC), a mammalian-cell optimized update to the CRAC protocol. In fCRAC, a fluorescent adaptor is used, in place of radiolabeling, to visualize RNA-protein complexes during gel-purification. fCRAC retains the tandem affinity purification and stringent, denaturing conditions of classical CRAC, enabling nucleotide-resolution mapping of protein:RNA interactions with high signal to noise ratio. We initially tested fCRAC using RPP25L, a component the RNase MRP and RNase P complexes. RPP25L almost exclusively bound to predicted, single sites in the RNA components (RMRP and RPPH1), showing excellent selectivity with nucleotide resolution. To support analysis of UV cross-linking data for more complex targets, we developed the trxtools package and example pipeline for standardized processing, quality control, and analysis of data from fCRAC and related methods. We include tailored strategies for repetitive RNA classes, such as tRNA and rRNA, which can be challenging to analyze using other approaches. We applied fCRAC and trxtools to define the RNA interactome of human CYCLON/CCDC86, a nuclear protein previously implicated in oncogenesis. This revealed specific interactions with rRNA, tRNA and ncRNAs involved in pre-rRNA and pre-tRNA processing. HighlightsO_LINucleotide-resolution definition of RNAs interacting with specific proteins, including rRNA and tRNA C_LIO_LIStringent denaturing purifications and robust visualization steps, with no requirement for radioactive labelling C_LIO_LITrxtools provides an integrated analysis pipeline with approaches for analyzing both single and multi-copy RNA species C_LI

The DEAD-box ATPase Dhh1 (DDX6 in humans) is a general activator of 5-3 mRNA decay that acts between the deadenylation and decapping steps of the pathway, although the exact mechanism of its action remains unclear. Dhh1 has been shown to interact with the MIF4G domain of the central scaffold protein of the Ccr4-Not deadenylase complex, Not1, as well as the decapping activator Edc3. Although structures have been published of Dhh1 in complex with Not1MIF4G or an Edc3 peptide, the impact of these interactions on the catalytic cycle of Dhh1 are unknown. Here, we show Edc3 enhances ATP and RNA binding by Dhh1, whereas Not1MIF4G promotes the catalytic step of ATP hydrolysis. Additionally, the modulation of Dhh1 activity by Edc3 requires a more extensive set of interaction motifs and interfaces than was previously recognized. While the effect of either Not1MIF4G or Edc3 on the ATPase activity of Dhh1 is modest, together both proteins increase Dhh1 activity over 200-fold, consistent with a role of Dhh1 in bridging 3 deadenylation and 5 decapping in the 5-3 mRNA decay pathway. These results suggest that Dhh1 coordinates deadenylation with decapping through changes in its ATP-coupled RNA binding affinity during its catalytic cycle. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/721454v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1369bf6org.highwire.dtl.DTLVardef@394ec5org.highwire.dtl.DTLVardef@6271e2org.highwire.dtl.DTLVardef@122cc6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Targeted knock-in technologies have enabled precise insertion of reporters, affinity tags, degrons, and other functional payloads into endogenous genomic loci. Over the past decade, a diverse collection of genome engineering strategies has emerged, including approaches based on homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), homology-mediated end joining (HMEJ), and related methodologies. While these advances have greatly expanded the capabilities of endogenous genome engineering, they have also increased the complexity of donor design, assembly, and validation. Here, we describe FORGE-KI (Functional Oncology Research Genetic Engineering - Knock in), a pathway-matched design workflow for endogenous knock-in engineering that aligns the assembly strategy with the underlying repair mechanism. For large-cargo insertions, we use a modular five-component framework that separates gene-specific targeting arms from reusable functional modules, allowing rapid assembly of HDR donor constructs targeting AHR, IRF1, and FOSL1 from a shared reagent collection. For MMEJ/PITCh applications, where short targeting elements permit rapid fabrication, we developed a streamlined one-step pipeline in which the entire donor and selection payload is synthesized as a single continuous fragment for direct cloning, compressing the design-to-reagent cycle time. This MMEJ workflow is paired with a dual-promoter nuclease vector (pForge-KI-MMEJ-Cas9-DualGuide) that drives the PITCh-release and locus-specific guides from distinct promoters, a design intended to reduce the repeated-promoter instability associated with some dual-guide vectors. We also established a standardized workflow for donor assembly, generation of knock-in cell populations, molecular validation, and selectable-cassette removal, and we demonstrate it by generating a functional, selection-marker-free, cytokine-inducible IRF1 HDR reporter line and an inducible IRF1 PITCh/MMEJ reporter pool with confirmed junction enrichment. In parallel, we developed forgeKI, an R package that automates C-terminal reporter knock-in design across both HDR and PITCh/MMEJ repair pathways, including guide selection, target-biology validation, targeting-arm design, domestication, donor-assembly planning, and generation of synthesis-ready constructs. Together, the reagents and software provide a practical system for endogenous knock-in engineering that supports multiple payloads, selection strategies, and repair pathways within a shared donor organization. Rather than replacing existing knock-in technologies, this framework provides a modular foundation for incorporating, extending, and automating the published knock-in methods.

Redox stress induces DNA mutations that contribute to chronic conditions affecting human health and to the emergence of antibiotic resistance. Yet, the impact of redox stress-induced mutagenesis remains difficult to decipher because redox agents are diverse and produce hard-to-detect mutational outcomes. Single-stranded DNA (ssDNA) provides a useful tool for studying mutagenic effects of redox agents, as it is particularly susceptible to damage and cannot be repaired by most DNA repair pathways. Here, we established a protocol to investigate redox stress-induced mutagenesis based on the Escherichia coli conjugative ssDNA that is transferred from donor to recipient cells. Using the environmentally relevant redox agents, potassium bromate and hydrogen peroxide, we show that the F episome is remarkably sensitive to weak mutagens during conjugation, enabling the detection of significant differences in mutational spectra induced by these agents. We support our findings with metabolomic analysis, which reveals agent-specific responses in E. coli. We compare these results with those obtained using a yeast ssDNA reporter and conclude that redox-induced mutagenesis depends, among other factors, on the metabolic context of the analysed system. These findings have important implications because the high sensitivity of conjugation-associated ssDNA to environmental mutagens may contribute to the evolution of antibiotic resistance. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/730102v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@14dfa25org.highwire.dtl.DTLVardef@61b5a8org.highwire.dtl.DTLVardef@dd4375org.highwire.dtl.DTLVardef@77f557_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA offers exceptional information density and stability, making it a promising medium for long-term data storage. However, the high cost of DNA synthesis and data retrieval remain key barriers to large-scale deployment. In this paper, we present DNA staples, a multipurpose library of short single-stranded DNA sequences that enables the encoding of arbitrary digital data by enzymatic assembly. Flexible encoding schemes allow the same presynthesized strand library to be used across applications, significantly reducing synthesis requirements while supporting diverse data representations. Using a restricted library also confers inherent error correction. In addition to storage, the library enables creation of computational DNA modules that perform highly parallel operations directly on stored data. This framework provides a cost-efficient approach to molecular data storage and supports integrated storage-computation at the DNA level.

The universally conserved enzyme uracil-DNA N-glycosylase (UNG) plays a central role in maintaining genome stability. It serves as the initiating factor in uracil base excision repair (UBER) by catalyzing the removal of uracil lesions in genomic DNA, a necessary first step in restoring genome integrity after hydrolytic deamination of cytosine to uracil or misincorporation of deoxy-uridine monophosphate during replication. Although methods have been developed to study UBER in vitro and in cellulo, none provide a quantitative readout of UNG activity on the chromosomal DNA of living cells. To address this gap, we created an UNG biosensor (U-report) that utilizes a modified cytosine base editor to generate a targeted genomic uracil lesion in a fluorescent reporter for C-to-U editing activity. UNG ablation through uracil DNA glycosylase inhibitor (Ugi) or UNG-knockout results in elevated reporter florescence. Surprisingly, isoform-specific knockouts reveal that mitochondrial UNG1 also contributes to UBER of nuclear DNA. Our studies combine to establish a real-time biosensor for quantification of chromosomal DNA uracil excision activity in living cells and indicate that both UNG isoforms should be considered in small molecule inhibitor development programs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/721890v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@bd2538org.highwire.dtl.DTLVardef@1d6b540org.highwire.dtl.DTLVardef@115aad4org.highwire.dtl.DTLVardef@182543d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Variants affecting RNA splicing are a major contributor to human disease, yet the consequences of variants outside of the canonical splice motifs are often difficult to determine. Here, we present a protocol for minigene-based evaluation of candidate splice-altering variants. The methodology described includes locus-specific insert design, commercial gene fragment synthesis, and long-read sequencing. The combined approach enables rapid assay development and nucleotide level resolution of the effect on splice isoforms in vitro, providing a scalable framework for functional validation of predicted cryptic splice variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/723105v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1a88cb5org.highwire.dtl.DTLVardef@adda98org.highwire.dtl.DTLVardef@1ea587corg.highwire.dtl.DTLVardef@574a63_HPS_FORMAT_FIGEXP M_FIG C_FIG

The MSH1 gene in Arabidopsis thaliana (AtMSH1) encodes a modular enzyme that consists of an N-terminal MutS DNA mismatch repair module fused to a C-terminal GIY-YIG nuclease. Disruption of MSH1 reverses the low organellar mutation rates that distinguish plants from other eukaryotes. However, the precise mechanism by which MSH1 prevents the accumulation of mutations remains unclear. Here, we show that AtMsh1 accurately recognizes and cleaves dsDNA containing mismatches and short indels. AtMsh1 efficiently cleaves dsDNA containing lesions generated by oxidative damage or deamination, with a strong preference for U:G mismatches. AtMsh1 cleaves DNA through an ATP-dependent enzymatic mechanism that requires divalent metal cofactors such as Mg2+. The enzyme introduces incisions at defined positions relative to the lesion or mismatch: approximately nine nucleotides 5' of the mismatch on the affected strand and twelve nucleotides 3' on the complementary strand. This offset cleavage generates staggered DNA ends with three-nucleotide overhangs. Although AtMsh1 displays positional specificity in its cleavage activity on substrates containing lesions and mismatches, it exhibits nonspecific double-stranded DNA cleavage in the presence of Mn2+. These findings establish AtMsh1 as a minimal mismatch repair (MMR) system in which mismatch/lesion recognition and DNA cleavage are functionally coupled. We propose that the resulting dsDNA breaks are processed by exonucleases that mediate single-stranded DNA resection, thereby removing the mismatch or lesion while generating a 3' single-stranded DNA overhang suitable for homologous recombination (HR) repair and gene conversion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/731605v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@156ce6eorg.highwire.dtl.DTLVardef@abb52org.highwire.dtl.DTLVardef@90286aorg.highwire.dtl.DTLVardef@4d7dce_HPS_FORMAT_FIGEXP M_FIG C_FIG Mismatch recognition and nuclease activity by plant organellar MutS Homolog 1 drive organellar genome maintenance.

Several eukaryotic mRNAs are polycistronic because of translatable open reading frames (ORFs) embedded in their untranslated regions (UTRs), in addition to the primary ORF (i.e., the coding sequence). While 5'UTR upstream ORFs (uORFs) are mechanistically and functionally well studied, 3'UTR downstream ORFs (dORFs) remain poorly understood. Here, we identify and characterize a short, translatable dORF in the 3'UTR of MAP3K10, which encodes a serine/threonine kinase involved in JNK signalling. A stringent computational screen predicted a conserved RNA G-quadruplex (rG4) within the 3'UTR of MAP3K10. Biophysical assays provided more evidence for rG4 formation, which drives translation of a conserved four-codon dORF. Disruption of the rG4 by point mutations or by an rG4-binding ligand reduced dORF translation. Reporter assays using constructs with strategically placed hairpin structures show that translation of the dORF is independent of both the 5 cap and the translation of the canonical ORF. Notably, deletion of the rG4-dORF module, either in exogenous constructs or in CRISPR-edited cells, led to reduced MAP3K10 expression. Together, these results provide evidence for the regulation of MAP3K10 expression by rG4-driven translation of a dORF. Thus, our study contributes to the growing body of evidence suggesting that dORF translation can regulate gene expression.

RNA plays a central role in the formation and regulation of biomolecular condensates, yet a quantitative understanding of how RNA sequence, structure, and thermodynamics jointly determine phase behaviour, particularly in repeat expansion RNAs, remains incomplete. Here, we introduce RNA2PS, a sequence-specific RNA coarse-grained model for phase-separation that predicts RNA structure from sequence, achieving quantitative agreement for both single-stranded conformations and duplex helical geometry relative to crystallographic PDB structures. RNA2PS represents each nucleotide by two beads that separate the phosphate-ribose backbone from the base. This representation decouples electrostatic interactions from directional base pairing, while explicitly incorporating strand polarity (5' [->] 3') and local sequence context at the trimer level. Canonical and wobble base pairing are modelled through a multi-body potential with sequence-dependent coordination. Importantly, RNA2PS captures sequence-dependent duplex stability at the nearest-neighbour level and reproduces experimental melting temperatures across a diverse set of sequences. RNA2PS shows that phase separation of trinucleotide repeat RNAs is governed by transient inter-strand duplexes that form reversible cross-links. Competition between intra- and intermolecular base pairing regulates the density of labile RNA-RNA interactions, giving rise to strong sequence- and length-dependent differences in condensation that reproduce cellular RNA foci formation. Overall, RNA2PS provides a near-quantitative predictive framework that links sequence-encoded hybridization thermodynamics to mesoscale condensation of pure RNA sequences.

DNA topoisomerase II (TOP2) generates transient DNA double-strand breaks that are trapped as TOP2-DNA covalent complexes (TOP2cc) by antibiotic and chemotherapy drugs. Here, we characterize tools for study of cellular responses to TOP2cc, exploiting a Saccharomyces cerevisiae TOP2 mutant (TOP2-F1025Y,R1128G) that generates spontaneous and inhibitor-induced covalent complexes at elevated frequencies. This Top2-hc (for "hypercleavage") mutant protein inhibits yeast cell growth when expressed alone or with endogenous Top2, and growth defects are exacerbated in DNA-repair-deficient genetic backgrounds and/or in the presence of low doses of the Top2 poison mAMSA. We generated analogous mutations in human and mouse TOP2A and TOP2B that gave increased TOP2cc, hypersensitization to topoisomerase poisons, increased DNA damage, and decreased cell survival in cultured cells. We further established knock-in mouse models with inducible, tissue-specific expression of each TOP2-hc isoform, demonstrating overt organismal toxicity and cellular markers of DNA damage responses. To illustrate the potential of these genetic tools, we carried out proof-of-principle screens in yeast and cultured human cells for sensitivity to TOP2-hc. The yeast screen revealed strong requirements for homologous recombination, moderate roles for sister chromatid cohesion and kinetochore function, and dependencies on vesicle and vacuolar functions. The pilot shRNA screen in human cells revealed shared requirements for resistance to expression of either TOP2A-hc or TOP2B-hc as well as examples of isoform specificity. These findings establish hypercleavage mutant proteins as effective tools for studying topoisomerase isoform-specific DNA damage and offer a foundation for exploring TOP2cc toxicity and tolerance in vivo. Significance statementDNA topoisomerase II enzymes untangle chromosomes by cutting DNA, but incomplete resealing creates toxic damage that is the basis of antibacterial and chemotherapy drugs. Here we describe toolkits in yeast, mammalian cells, and mice that take advantage of mutant topoisomerase II enzymes that trap on DNA without drugs, creating powerful genetic systems to better study how cells deal with this type of DNA damage. We provide benchmarking data to validate these tools and to illustrate how they can be used for screens in cultured cells or tissue-specific experiments in vivo. These toolkits overcome longstanding technical barriers and enable new ways to study topoisomerase II-mediated DNA damage.

Existing databases of interphase chromosome conformations typically store three-dimensional coordinates of genomic segments. However, since interphase chromatin is highly dynamic, such databases are dominated by transient configurations and unstructured regions, whose positions vary continuously between cells and over time, unlike folded proteins such as globin, which adopt similar structures in every cell. These drawbacks motivated the inception of a database based on strion (a portmanteau of a string capturing structure and function). A strion concisely describes the structure and activity of all transcription units in one cell, by retaining only functionally relevant positional information. Sets of strions describing structures in different cells sampled at different times are compiled into a super-strion. Then, 46 super-strions summarise the range of structure and activity of a human cell type, including information on all transcription units, how often each co-fires and co-clusters with others in transcription factories/hubs, enhancer interactomes and small-world expression networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/724942v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13a1263org.highwire.dtl.DTLVardef@18d2c78org.highwire.dtl.DTLVardef@162865corg.highwire.dtl.DTLVardef@1631d65_HPS_FORMAT_FIGEXP M_FIG C_FIG