Nucleic Acids Research最新文献

Time-resolved small-angle X-ray experiments are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in the folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 24.3 to 12.6 Å. The collapse is monophasic and is complete in <600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, showing that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favour excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single-stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that Bxb1 RDF is probably an exonuclease and TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein-protein interaction at the junction of recombinase's second DNA binding domain and the base of the coiled-coil domain.

Large vertebrate genomes duplicate by activating tens of thousands of DNA replication origins, irregularly spaced along the genome. The spatial and temporal regulation of the replication process is not yet fully understood. To investigate the DNA replication dynamics, we developed a methodology called RepliCorr, which uses the spatial correlation between replication patterns observed on stretched single-molecule DNA obtained by either DNA combing or high-throughput optical mapping. The analysis revealed two independent spatiotemporal processes that regulate the replication dynamics in the Xenopus model system. These mechanisms are referred to as a fast and a slow replication mode, differing by their opposite replication fork speed and rate of origin firing. We found that Polo-like kinase 1 (Plk1) depletion abolished the spatial separation of these two replication modes. In contrast, neither replication checkpoint inhibition nor Rap1-interacting factor (Rif1) depletion affected the distribution of these replication patterns. These results suggest that Plk1 plays an essential role in the local coordination of the spatial replication program and the initiation-elongation coupling along the chromosomes in Xenopus, ensuring the timely completion of the S phase.

Hypoxia enhances histone methylation by inhibiting oxygen- and α-ketoglutarate-dependent demethylases, resulting in increased methylated histones. This study reveals how hypoxia-induced methylation affects histone clipping and the reorganization of heterochromatin into senescence-associated heterochromatin foci (SAHF) during oncogene-induced senescence (OIS) in IMR90 human fibroblasts. Notably, using top-down proteomics, we discovered specific cleavage sites targeted by Cathepsin L (CTSL) in H3, H2B and H4 during Raf activation, identifying novel sites in H2B and H4. Hypoxia counteracts CTSL-mediated histone clipping by promoting methylation without affecting CTSL's activity. This increase in methylation under hypoxia protects against clipping, reshaping the epigenetic landscape and influencing chromatin accessibility, as shown by ATAC-seq analysis. These insights underscore the pivotal role of hypoxia-induced histone methylation in protecting chromatin from significant epigenetic shifts during cellular aging.

Most eukaryotic genomes are transcribed pervasively, thereby producing an array of long non-coding RNAs (lncRNAs) in addition to protein-coding mRNAs. A large fraction of these lncRNAs is targeted by polyadenylation-dependent decay via the poly(A)-binding protein nuclear 1 (PABPN1) and the RNA exosome. Yet, how PABPN1 contributes to nuclear RNA surveillance by facilitating lncRNA turnover by the RNA exosome remains largely unclear. Here, we show that PABPN1 is important for the nuclear retention of polyadenylated lncRNAs, such that PABPN1 loss of function allows target lncRNAs to evade nuclear decay, leading to cytoplasmic accumulation. Interestingly, we found that another nuclear PABP, ZC3H14, functions antagonistically to PABPN1 and the poly(A)-tail exosome targeting (PAXT) connection in the control of nuclear lncRNA turnover. Collectively, our findings disclose the critical interplay between two conserved nuclear PABPs, PABPN1 and ZC3H14, in RNA surveillance via the control of nuclear RNA export.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGOs) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotically-arrested NGOs. In male germ cells, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes, allowing germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

Single-stranded-double-stranded DNA (ss-dsDNA) replication forks and primer-template junctions are important recognition sites for the assembly and function of proteins involved in DNA replication, recombination, and repair. DNA 'breathing' - i.e. thermally induced local fluctuations of the sugar-phosphate backbones and bases - can populate metastable conformational macrostates at positions near such junctions and likely play key roles in the functional interactions of the regulatory proteins that bind at these sites. Recently, Maurer et al. [1] performed polarization-sweep single-molecule fluorescence (PS-SMF) studies on exciton-coupled (iCy3)2 dimer-labeled ss-dsDNA fork constructs, which revealed that the nucleobases and backbones immediately adjacent to the dimer probes undergo conformational fluctuations on time scales ranging from hundreds of microseconds to hundreds of milliseconds. The local conformations sensed by the dimer probes consist of four quasi-stable macrostates whose populations and dynamics depend on dimer probe position relative to these junctions. Here we present theoretical analyses of these PS-SMF data that quantify the relative stabilities and activation barriers of the free energy surfaces of site-specific DNA 'breathing' events at key positions within these junctions. Our results suggest a detailed molecular picture for DNA 'breathing' at these positions, thus providing insights into understanding the molecular mechanisms of the proteins that operate at these sites.

Bacterial pathogens, such as Yersinia pseudotuberculosis, encounter temperature fluctuations during host infection and upon return to the environment. These temperature shifts impact RNA structures globally. While previous transcriptome-wide studies have focused on RNA thermometers in the 5'-untranslated region of virulence-related messenger RNAs, our investigation revealed temperature-driven structural rearrangements in the small RNA CyaR (cyclic AMP-activated RNA). At 25°C, CyaR primarily adopts a conformation that occludes its seed region, but transitions to a liberated state at 37°C. By RNA sequencing and in-line probing experiments, we identified the Shine-Dalgarno sequence of ompX as a direct target of CyaR. Interestingly, the ompX transcript itself exhibits RNA thermometer-like properties, facilitating CyaR base pairing at elevated temperatures. This interaction impedes ribosome binding to ompX and accelerates degradation of the ompX transcript. Furthermore, we observed induced proteolytic turnover of the OmpX protein at higher temperatures. Collectively, our study uncovered multilayered post-transcriptional mechanisms governing ompX expression, resulting in lower OmpX levels at 37°C compared with 25°C.