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Trends in Biochemical Sciences Jun 2024Interactions of RNA with DNA are principles of gene expression control that have recently gained considerable attention. Among RNA-DNA interactions are R-loops and... (Review)
Review
Interactions of RNA with DNA are principles of gene expression control that have recently gained considerable attention. Among RNA-DNA interactions are R-loops and RNA-DNA hybrid G-quadruplexes, as well as RNA-DNA triplexes. It is proposed that RNA-DNA triplexes guide RNA-associated regulatory proteins to specific genomic locations, influencing transcription and epigenetic decision making. Although triplex formation initially was considered solely an in vitro event, recent progress in computational, biochemical, and biophysical methods support in vivo functionality with relevance for gene expression control. Here, we review the central methodology and biology of triplexes, outline paradigms required for triplex function, and provide examples of physiologically important triplex-forming long non-coding RNAs.
Topics: DNA; RNA; Humans; Animals; Nucleic Acid Conformation
PubMed: 38582689
DOI: 10.1016/j.tibs.2024.03.009 -
Hereditas May 2024
Topics: DNA; Humans; History, 21st Century; History, 20th Century
PubMed: 38711122
DOI: 10.1186/s41065-024-00319-x -
Nature Dec 2023FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity....
FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain-a DNA-binding domain that is commonly thought to function as a monomer or dimer-to form a higher-order multimer after binding to TG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with TG repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two 'side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a 'rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.
Topics: Base Sequence; Consensus Sequence; Cryoelectron Microscopy; DNA; Forkhead Transcription Factors; Microsatellite Repeats; Mutation; Nucleotide Motifs; Protein Domains; Protein Multimerization; T-Lymphocytes, Regulatory
PubMed: 38030726
DOI: 10.1038/s41586-023-06793-z -
Cell Reports May 2024Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We...
Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.
Topics: DNA Replication; Humans; Ubiquitination; DNA Repair; DNA
PubMed: 38703364
DOI: 10.1016/j.celrep.2024.114178 -
Nature Structural & Molecular Biology Sep 2023SUMOylation regulates numerous cellular processes, but what represents the essential functions of this protein modification remains unclear. To address this, we...
SUMOylation regulates numerous cellular processes, but what represents the essential functions of this protein modification remains unclear. To address this, we performed genome-scale CRISPR-Cas9-based screens, revealing that the BLM-TOP3A-RMI1-RMI2 (BTRR)-PICH pathway, which resolves ultrafine anaphase DNA bridges (UFBs) arising from catenated DNA structures, and the poorly characterized protein NIP45/NFATC2IP become indispensable for cell proliferation when SUMOylation is inhibited. We demonstrate that NIP45 and SUMOylation orchestrate an interphase pathway for converting DNA catenanes into double-strand breaks (DSBs) that activate the G2 DNA-damage checkpoint, thereby preventing cytokinesis failure and binucleation when BTRR-PICH-dependent UFB resolution is defective. NIP45 mediates this new TOP2-independent DNA catenane resolution process via its SUMO-like domains, promoting SUMOylation of specific factors including the SLX4 multi-nuclease complex, which contributes to catenane conversion into DSBs. Our findings establish that SUMOylation exerts its essential role in cell proliferation by enabling resolution of toxic DNA catenanes via nonepistatic NIP45- and BTRR-PICH-dependent pathways to prevent mitotic failure.
Topics: DNA, Catenated; Anaphase; DNA; Sumoylation
PubMed: 37474739
DOI: 10.1038/s41594-023-01045-0 -
Nucleus (Austin, Tex.) Dec 2024DNA double-strand break (DSB) is the most dangerous type of DNA damage, which may lead to cell death or oncogenic mutations. Homologous recombination (HR) and... (Review)
Review
DNA double-strand break (DSB) is the most dangerous type of DNA damage, which may lead to cell death or oncogenic mutations. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two typical DSB repair mechanisms. Recently, many studies have revealed that liquid-liquid phase separation (LLPS) plays a pivotal role in DSB repair and response. Through LLPS, the crucial biomolecules are quickly recruited to damaged sites with a high concentration to ensure DNA repair is conducted quickly and efficiently, which facilitates DSB repair factors activating downstream proteins or transmitting signals. In addition, the dysregulation of the DSB repair factor's phase separation has been reported to promote the development of a variety of diseases. This review not only provides a comprehensive overview of the emerging roles of LLPS in the repair of DSB but also sheds light on the regulatory patterns of phase separation in relation to the DNA damage response (DDR).
Topics: DNA Breaks, Double-Stranded; Phase Separation; DNA Repair; Homologous Recombination; DNA
PubMed: 38146123
DOI: 10.1080/19491034.2023.2296243 -
Biochimie Nov 2023Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial biological activities. Among these oddities, guanine-rich DNA sequences... (Review)
Review
Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial biological activities. Among these oddities, guanine-rich DNA sequences can form unusual four-stranded secondary structures called G-quadruplexes (G4s). G4-prone sequences have been found in the genomes of most species, and G4s play important roles in essential processes such as transcription, replication, genome integrity and epigenetic regulation. Here, we present a short overview of G-quadruplexes followed by a detailed description of the biophysical and biochemical methods used to characterize G4s in vitro. The principles, experimental details and possible shortcomings of each method are discussed to provide a comprehensive view of the techniques used to study these structures. We aim to provide a set of guidelines for standardizing research on G-quadruplexes; these guidelines are not meant to be a dogmatic set of rules, but should rather provide useful information on the methods currently used to study these fascinating motifs.
Topics: G-Quadruplexes; Epigenesis, Genetic; DNA; Genome
PubMed: 36596406
DOI: 10.1016/j.biochi.2022.12.019 -
The EMBO Journal Sep 2023The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we...
The conserved protein HMCES crosslinks to abasic (AP) sites in ssDNA to prevent strand scission and the formation of toxic dsDNA breaks during replication. Here, we report a non-proteolytic release mechanism for HMCES-DNA-protein crosslinks (DPCs), which is regulated by DNA context. In ssDNA and at ssDNA-dsDNA junctions, HMCES-DPCs are stable, which efficiently protects AP sites against spontaneous incisions or cleavage by APE1 endonuclease. In contrast, HMCES-DPCs are released in dsDNA, allowing APE1 to initiate downstream repair. Mechanistically, we show that release is governed by two components. First, a conserved glutamate residue, within HMCES' active site, catalyses reversal of the crosslink. Second, affinity to the underlying DNA structure determines whether HMCES re-crosslinks or dissociates. Our study reveals that the protective role of HMCES-DPCs involves their controlled release upon bypass by replication forks, which restricts DPC formation to a necessary minimum.
Topics: DNA; Proteins; DNA Damage; DNA, Single-Stranded; DNA Repair
PubMed: 37519246
DOI: 10.15252/embj.2022113360 -
Nucleic Acids Research Aug 2023In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day... (Review)
Review
In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.
Topics: DNA; Formaldehyde; Paraffin Embedding; Sequence Analysis, DNA; Tissue Fixation
PubMed: 37351572
DOI: 10.1093/nar/gkad519 -
Bioinformatics (Oxford, England) Nov 2023The two strands of the DNA double helix locally and spontaneously separate and recombine in living cells due to the inherent thermal DNA motion. This dynamics results in...
MOTIVATION
The two strands of the DNA double helix locally and spontaneously separate and recombine in living cells due to the inherent thermal DNA motion. This dynamics results in transient openings in the double helix and is referred to as "DNA breathing" or "DNA bubbles." The propensity to form local transient openings is important in a wide range of biological processes, such as transcription, replication, and transcription factors binding. However, the modeling and computer simulation of these phenomena, have remained a challenge due to the complex interplay of numerous factors, such as, temperature, salt content, DNA sequence, hydrogen bonding, base stacking, and others.
RESULTS
We present pyDNA-EPBD, a parallel software implementation of the Extended Peyrard-Bishop-Dauxois (EPBD) nonlinear DNA model that allows us to describe some features of DNA dynamics in detail. The pyDNA-EPBD generates genomic scale profiles of average base-pair openings, base flipping probability, DNA bubble probability, and calculations of the characteristically dynamic length indicating the number of base pairs statistically significantly affected by a single point mutation using the Markov Chain Monte Carlo algorithm.
AVAILABILITY AND IMPLEMENTATION
pyDNA-EPBD is supported across most operating systems and is freely available at https://github.com/lanl/pyDNA_EPBD. Extensive documentation can be found at https://lanl.github.io/pyDNA_EPBD/.
Topics: Computer Simulation; Models, Chemical; DNA; Software; Base Pairing; Nucleic Acid Conformation
PubMed: 37991847
DOI: 10.1093/bioinformatics/btad699