Nucleic Acids Research

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is a key enzyme in the Base Excision Repair pathway, responsible for processing abasic (AP-) sites. Recent studies revealed that APE1 participates in repairing DNA secondary structures as G-quadruplexes (G4). Telomeres, stabilized by shelterin proteins, are rich in G4, where APE1 binds and repairs AP-sites to maintain their integrity. The complementary cytosine-rich strand forms another structure, the i-motif (iM), essential for telomere maintenance, though its repair mechanism remains unclear. Herein we investigate APE1 binding and processing capabilities toward native and damaged telomeric iM, bearing AP-sites in different positions. Using biochemical and biophysical assays, we found that APE1 binds the telomeric iM-sequence and that its cleavage efficiency depends on AP-site position within iM. Proximity Ligation Assay analysis, in HeLa and U2OS cells, highlighted a novel interaction between APE1 and PCBP1, a well-known iM-folding modulator. PCBP1 binds iM with higher affinity than APE1 and inhibits its cleavage activity on damaged iM. Immunofluorescence and Telomere Restriction Fragment analyses showed that depletion of APE1 or PCBP1 impairs their interaction with the shelterin components, affecting telomere length. These results connect APE1 canonical DNA repair activity with the maintenance of non-canonical DNA secondary structures in telomeres, through its interaction with PCBP1. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/694817v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1962c63org.highwire.dtl.DTLVardef@3c3f60org.highwire.dtl.DTLVardef@164d78borg.highwire.dtl.DTLVardef@1831aae_HPS_FORMAT_FIGEXP M_FIG C_FIG

Withdrawal StatementThe authors have withdrawn this manuscript because they identified problems with how some figure panels were processed. Those experiments will be repeated before deposition of a new manuscript. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors.

Intricate interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. UV crosslinking methods to study protein-RNA interactions have been instrumental in elucidating their interactions but can be limited by degradation of target proteins during the process, low signal-to-noise ratios, and non-specific interactions. Addressing these limitations, we describe reCRAC (reverse CRAC), a novel adaptation of the CRAC (crosslinking and analysis of cDNA) technique, optimized for yeast Saccharomyces cerevisiae. Like CRAC, reCRAC applies tandem affinity purification to yield highly enriched protein preparations. However, reCRAC is redesigned to work with unstable proteins. This is achieved by lysing the cells directly into highly denaturing buffer conditions, followed by stringent purification steps. The reCRAC method was successfully applied to the easily degraded yeast protein Pin4, allowing identification of precise binding sites at base-pair resolution with greatly reduced target protein degradation and improved signal-to-noise ratios.

DNA copy number changes are the most frequent genomic alterations in cancer cells. The ribosomal DNA (rDNA) region is particularly vulnerable to such changes due to its repetitive nature. Here, we demonstrate that Rad27/FEN-1, a structure-specific nuclease in budding yeast, plays a crucial role in maintaining rDNA stability. The production of extrachromosomal rDNA circles and severe chromosomal rDNA instability are observed in the rad27{Delta} mutant, independently of Fob1-mediated DNA replication fork arrest and DNA double-strand break (DSB) formation in the rDNA. The rad27{Delta} mutant accumulates unprocessed Okazaki fragments in the rDNA region, without inducing DSB formation. Similar rDNA instability is observed in DNA ligaseCdc9-deficient cells. Furthermore, we show that Exonuclease 1 and PCNA can compensate for the loss of Rad27 function in the rDNA stabilization. These findings highlight the importance of proper Okazaki fragment processing in preventing non-DSB-induced rDNA copy number changes.

One challenge faced by scientists from the alternative RNA splicing field is to decode the cooperative or antagonistic effects of splicing factors to understand and eventually predict splicing outcomes on a genome-wide scale. In this manuscript, we introduce SplicingLore, an open access database and web resource that help to fill this gap in a straightforward manner. The database contains a collection of RNA-seq-derived lists of alternative exons regulated by a total of 75 different splicing factors. All datasets were processed in a standardized manner, ensuring valid comparisons and correlation analyses. The user can easily retrieve a factor-specific set of differentially included exons from the database, or provide a list of exons and search which splicing factor(s) control(s) their inclusion. Our simple workflow is fast and easy to run, and it ensures a reliable calculation of correlation scores between the tested datasets. As a proof of concept, we predicted and experimentally validated a novel functional cooperation between the RNA helicases DDX17 and DDX5 and the HNRNPC protein. SplicingLore is available at https://splicinglore.ens-lyon.fr/.

This study identifies a novel operon-driven signaling module in Deinococcus radiodurans. The operon includes a von Willebrand A domain protein (DRA0331), a Ser/Thr protein kinase (DRA0332), a canonical FHA-domain protein (DRA0333), and a PP2C-type phosphatase (DRA0334). DRA0334 shows Mn{superscript 2}{square}-dependent phosphatase activity and has a unique dual-domain structure that combines a Kinase-Interacting FHA (KI-FHA) domain with a PP2C catalytic domain. Functional assays show that FHA-domain protein, DRA0333 boosts the phosphorylation of STPKs like RqkA and DR1243 while operonic partner PP2C-type phosphatase, DRA0334 counteracts this through targeted dephosphorylation, establishing a phospho-regulatory antagonism. Notably, the KI-FHA domain of the DRA0334 phosphatase competitively interacts with the FHA domain to modulate the radiation-responsive RqkA kinase, thereby maintaining kinase-phosphatase balance. This KI-FHA domain also imparts substrate specificity and enables feedback regulation. Additionally, DRA0334 modular variants confirm separate roles of catalytic and docking modules, and STRING analyses link DRA0334 functions to DNA repair and stress recovery. Collectively, the findings suggest an operonic connection between DRA0333 and DRA0334, indicating that the KI-FHA and FHA domains may act as phospho-docking switches. These switches can regulate both kinase and phosphatase activities in a push-pull regulatory mechanism within the phosphorylation-dephosphorylation cycle of signal transduction, depending on their association with the type of catalytic domain.

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of [~]20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5'-to-3' direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

The segregation of bacterial chromosomes is widely mediated by partitioning proteins (ParAB). While ParB binds DNA specifically by recognising short, palindromic sequences known as parS sites, ParA utilises its ATPase activity to generate the force to translocate ParB-DNA nucleoprotein complexes (segrosomes). The assembly of the segrosome requires the association of ParB with parS, followed by nonspecific spread of the protein along the DNA. To spread on DNA, the ParB dimer must entrap the parS site within the complex, a process triggered by CTP binding to the conserved GERR amino acid motif. In Streptomyces, a genus of soil-dwelling, multigenomic bacteria that have a complex life cycle, ParB-dependent chromosome partitioning is initiated during the growth of sporogenic hyphae. However, the molecular mechanisms underlying segrosome formation in Streptomyces and their ability to coordinate with sporogenic development remain incompletely understood. In this study, we advance the understanding of chromosome segregation in bacteria by exploring the effects of CTP binding and hydrolysis on the formation of the partitioning complex in S. coelicolor. Here, via in vitro approaches, we demonstrate that a conserved GERR motif is essential for CTP binding and hydrolysis by S. coelicolor ParB. Moreover, the motif is crucial for CTP-dependent ParB accumulation on DNA. Using mutant strains, we show the significance of the GERR motif for segrosome complex assembly. Additionally, we provide data showing that the CTP-binding motif contributes to the regulation of the growth of sporogenic cells. Overall, we show that CTP-dependent segrosome assembly impacts the development of S. coelicolor sporogenic cells.

The Saccharomyces cerevisiae Irc3 protein is a mitochondrial Superfamily II DNA helicase that, according to genomic data, is conserved in different yeast species. Here we characterize Irc3 helicase from the thermotolerant yeast Ogataea polymorpha (Irc3op) that throughout its helicase motor domain has approximately 82% similarity with the S. cerevisiae protein. Irc3op retains enzymatic activity at considerably higher temperatures than Irc3sc, displaying the fastest rate of ATP hydrolysis at 41 {degrees}C and a Tm of 45.3 {degrees}C for its secondary structure melting. We demonstrate that Irc3op is a structure-specific DNA helicase translocating on both single- and double-stranded DNA molecules. Like the homolog of S. cerevisiae, Irc3op can unwind only DNA molecules that contain branched structures. Different DNA molecules containing three- and four-way branches are utilized by Irc3op as unwinding substrates. Importantly, the preferred unwinding substrate of Irc3op is a DNA fork containing the nascent lagging strand, suggesting a possible role in replication restart following a block in leading strand polymerization. A lower unwinding efficiency of four-way branched DNA molecules could explain why Irc3op only partially complements the irc3{Delta} phenotype in S. cerevisiae.

R-loops exert varied beneficial or detrimental effects. To assess the function of an R-loop at a specific genetic locus, we had developed an inducible RNaseH1-EGFP-dCas9 (RED) protein chimaera as part of a locus-associated R-loop-modulating system (LasR). LasR is compatible with R-loop modulation in trans, which targets RED to one locus to repress R-loops at another spatially proximal site. Here we use the LasR system for R-loop modulation in cis, which consists of targeting RED directly to an R-loop. The combination of LasR in cis and trans will be essential to ascribe functions to specific R-loops within varied molecular contexts and study designs.

The Rev1 deoxycytidyl transferase functions as a scaffold protein for DNA polymerase {zeta} (Pol {zeta})-mediated translesion synthesis (TLS). Biochemical studies with yeast enzymes indicate that Rev1 plays a dual regulatory role in TLS, stimulating Pol {zeta} activity at sites of damage but inhibiting its activity on undamaged DNA. An evolutionary conserved N-terminal alpha-helical motif (M1), located 10-20 amino acids upstream of Rev1s single BRCT domain, is required for the inhibitory activity of Rev1 on undamaged DNA. Mutations in the M1 motif result in a stimulation of Pol {zeta} replication activity on both undamaged and damaged DNA. Yeast cells carrying a REV1 mutant lacking the M1 motif, show a significant increase in mutation track length, without significantly affecting overall spontaneous mutation rates. The regulatory activity of Rev1 is independent of its catalytic activity. However, it requires that Rev1-Pol {zeta} is a stable complex, and that this complex is coordinated by the replication clamp PCNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/700666v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@9b40dorg.highwire.dtl.DTLVardef@10bf5d1org.highwire.dtl.DTLVardef@376ff4org.highwire.dtl.DTLVardef@1970db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutieres syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS-ortholog mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these yeast AGS-ortholog mutants. We found higher rNMP incorporation in the nuclear genome of rnh201-G42S than in wild-type and rnh203-K46W-mutant cells, and an elevated rCMP content in both mutants. Moreover, we uncovered unique rNMP patterns in each mutant, highlighting a differential activity of the AGS mutants towards rNMPs embedded on the leading or on the lagging strand of DNA replication. This study guides future research on rNMP characteristics in human genomic samples carrying AGS mutations.

Most known modification-dependent restriction endonucleases target 5-methylcytosine, only a few N6-methyladenine (6mA)-dependent restriction endonucleases have been well-characterized, and the majority of them recognize the G6mATC motif (e.g., DpnI, HHPV4I). Here, we report the identification of a novel 6mA-dependent DNA-binding protein from Vibrio cholerae, VchI, which specifically recognizes the G6mAG motif. VchI contains a winged helix (wH) domain that is homologous to the wH domain in DpnI. However, several key residues involved in 6mA recognition differ between VchI and DpnI, which may contribute to the discrepancy in their recognition specificities. These findings advance our understanding of prokaryotic 6mA modification diversity and the 6mA recognition mechanism of the wH domain, while simultaneously providing an innovative tool for epigenetic research.

The identification of miRNA targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily translated to an in vivo setting. To overcome these limitations and to facilitate the investigation of miRNA functions in mice, we have developed a method (HEAP: for Halo-Enhanced Ago2 Pulldown) to map miRNA-mRNA binding sites. This method is based on a novel genetically engineered mouse harboring a conditional, Cre-regulated, Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be efficiently purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, in developing embryos, in adult tissues and in autochthonous mouse models of human brain and lung cancers.\n\nThe tools and the datasets we have generated will serve as a valuable resource to the scientific community and will facilitate the characterization of miRNA functions under physiological and pathological conditions.

Integrator is a multi-subunit complex that directly interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Through its RNA endonuclease activity, Integrator is required for 3'-end processing of both non-coding and coding transcripts. Here we demonstrate that depleting Integrator subunit 11 (INTS11), the main catalytic subunit of the Integrator complex, leads to a global elongation defect as a result of decreased polymerase processivity. We observe this defect in the region approximately 12 to 35 kb downstream of the transcription start site (TSS), where RNAPII normally transitions to its maximum processivity. We also identify an important role for INTS11, possibly in association with RNAPII CTD phospho-Tyr1, in repressing antisense transcription upstream of active promoters, as well as repressing transcription of genic regions near AsiSI-induced double-strand breaks. Altogether, this study points toward a novel function of Integrator in promoting termination of incompetent RNAPII molecules while facilitating the transition to fully processive polymerase in order to enable efficient elongation.

Genes do not act in isolation but through complex networks of interactions. While many double and triple gene interactions have been uncovered in Saccharomyces cerevisiae, most have been identified through overexpression or loss-of-function studies, leaving dosage-dependent interactions largely unexplored. Multi-gene control systems suffer from various drawbacks, including limited range and high cell-to-cell variation of expression across the population. To address this gap, we extend a recently developed inducible expression system that enables precise, tunable control of gene expression to additional repressor-inducer pairs: LacI/IPTG and LexA-hER/{beta}-estradiol. We demonstrate that both new systems can control endogenous genes on their own by placing various low, medium and high expressed endogenous genes under their control. Using all three WTC systems, we show simultaneous and orthogonal double and triple gene control that confirms known gene-gene interactions within the anaphase signaling network and reveals new dosage-dependent phenotypes. Given their precision, orthogonality and compatibility with all commonly used growth media, this collection of WTC systems represents a new tool suitable for precise investigation of dosage-dependent gene-gene interactions in yeast.

DNA ligases, essential enzymes which re-join the backbone of DNA come in two structurally-distinct isoforms, NAD-dependent and ATP-dependent, which differ in cofactor usage. The present view is that all bacteria exclusively use NAD-dependent DNA ligases for DNA replication, while archaea and eukaryotes use ATP-dependent DNA ligases. Some bacteria also possess auxiliary ATP-dependent DNA ligases; however, these are only employed for specialist DNA repair processes. Here we show that in the genomes of high-light strains of the marine cyanobacterium Prochlorococcocus marinus, an ATP-dependent DNA ligase has replaced the NAD-dependent form, overturning the present paradigm of a clear evolutionary split in ligase usage. Genes encoding partial NAD-dependent DNA ligases are found on mobile regions in highlight genomes and lack domains required for catalytic function. This constitutes the first reported example of a bacterium that relies on an ATP-dependent DNA ligase for DNA replication and recommends P. marinus as a model to investigate the evolutionary origins of these essential DNA-processing enzymes.

There are significant differences in the components of the ribosomal DNA transcription apparatuses of yeast and mammals. Moreover, the patterns of regulation between mammals and yeast are also different. To overcome, deficits in our understanding of mammalian rDNA transcription, we have developed a system to introduce an inducible degron into the endogenous genes of mammalian cells. This allows us to combine a knock out the endogenous gene product and replace it with mutant proteins in order to study their function in ribosomal DNA transcription. Using this system, we show that the knockout of PAF49, the mammalian ortholog of yeast A34, results in the relatively rapid degradation of PAF53, the ortholog of yeast A49. Interestingly, the steady-state levels of the core subunits of RNA polymerase I are unaffected.

Time-resolved transcriptomic profiling has been used to study phage-host interactions for more than a decade. However, the resulting datasets are not readily accessible for custom re-analysis, and resources are lacking that provide standardized processing, storage, and analysis of transcriptomes from phage infections. Here, we present the PhageExpressionAtlas, the first bioinformatics resource for storing time-resolved dual RNA-sequencing data from phage infections. This data was processed uniformly using a custom analysis pipeline and is presented for interactive exploration through visualisation. The PhageExpressionAtlas currently hosts 42 datasets from 23 studies. Using the PhageExpressionAtlas, we replicate key findings from original publications and extend hypothesis testing across multiple phage-host systems. By systematically querying and analyzing the underlying database, we evaluate approaches to phage gene classification and show that uncharacterized phage genes are expressed across all infection phases. Moreover, we provide a comprehensive view of the expression dynamics of anti-phage defenses as well as host- and phage-encoded anti-defense systems in the infection context, indicating unique and conserved patterns of transcriptional regulation underlying bacterial anti-phage immunity and phage counter-strategies. Together, the PhageExpressionAtlas is a unifying resource that democratizes transcriptomics-driven analyses of phage-host interactions and supports integrative cross-study assessment.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBL{Delta}C25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBL{Delta}C25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBL{Delta}C25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.