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Molecular Cell Jul 2023Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that...
Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.
Topics: Transcription Factors; RNA; Binding Sites; Protein Binding; DNA
PubMed: 37402367
DOI: 10.1016/j.molcel.2023.06.012 -
Molecular Cell Aug 2023Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF...
Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.
Topics: CCCTC-Binding Factor; R-Loop Structures; Chromatin; Cell Cycle Proteins; DNA; Cohesins
PubMed: 37536339
DOI: 10.1016/j.molcel.2023.07.006 -
Immunity Nov 2023Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm...
Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm and initiates innate immune responses and has been implicated in various infectious and non-infectious contexts. cGAS binds to the backbone of dsDNA and generates the second messenger, cGAMP, which activates the stimulator of interferon genes (STING). Here, we show that the endogenous polyamines spermine and spermidine attenuated cGAS activity and innate immune responses. Mechanistically, spermine and spermidine induced the transition of B-form DNA to Z-form DNA (Z-DNA), thereby decreasing its binding affinity with cGAS. Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism that decreases the cellular concentrations of spermine and spermidine, enhanced cGAS activation by inhibiting cellular Z-DNA accumulation; SAT1 deficiency promoted herpes simplex virus 1 (HSV-1) replication in vivo. The results indicate that spermine and spermidine induce dsDNA to adopt the Z-form conformation and that SAT1-mediated polyamine metabolism orchestrates cGAS activity.
Topics: Spermine; Spermidine; DNA, Z-Form; DNA, B-Form; DNA; Nucleotidyltransferases; Polyamines; Immunity, Innate
PubMed: 37848037
DOI: 10.1016/j.immuni.2023.09.012 -
Science (New York, N.Y.) Aug 2023Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage...
Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded-single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5' end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a "POT-hole" surface, the mutation of which compromises junction protection in vitro and telomeric 5'-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a's sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded-single-stranded junction.
Topics: Animals; Humans; Mice; Crystallography; DNA; Mutation; Shelterin Complex; Telomere; Telomere-Binding Proteins
PubMed: 37590346
DOI: 10.1126/science.adi2436 -
Journal of Materials Chemistry. B Aug 2023DNA has excellent molecular recognition properties. At the same time, DNA has a programmable structure, high stability, and can be easily modified, making DNA attractive... (Review)
Review
DNA has excellent molecular recognition properties. At the same time, DNA has a programmable structure, high stability, and can be easily modified, making DNA attractive for biosensor design. To convert DNA hybridization or aptamer binding events to physically detectable signals, various nanomaterials have been extensively exploited to take advantage of their optical and surface properties. A popular sensing scheme is through the adsorption of a fluorescently-labeled DNA probe, where detection is achieved by target-induced probe desorption and fluorescence recovery. Another method is to use DNA to protect the colloidal stability of nanomaterials, where subsequent target binding can decrease the protection ability and induce aggregation; this method has mainly been used for gold nanoparticles. This Perspective summarizes some of our work in examining the sensing mechanisms, and we articulate the importance of the understanding of DNA/surface and target/surface interactions for the development of practical DNA-based biosensors.
Topics: Gold; Metal Nanoparticles; DNA; Biosensing Techniques; Nanostructures
PubMed: 37465903
DOI: 10.1039/d3tb01100c -
Accounts of Chemical Research May 2024In 1960, Weber prophesied that "There are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and...
In 1960, Weber prophesied that "There are many ways in which the properties of the excited state can be utilized to study points of ignorance of the structure and function of proteins". This has been realized, illustrating that an intrinsic and highly responsive fluorophore such as tryptophan can alter the course of an entire scientific discipline. But what about RNA and DNA? Adapting Weber's protein photophysics prophecy to nucleic acids requires the development of intrinsically emissive nucleoside surrogates as, unlike Trp, the canonical nucleobases display unusually low emission quantum yields, which render nucleosides, nucleotides, and oligonucleotides practically dark for most fluorescence-based applications.Over the past decades, we have developed emissive nucleoside surrogates that facilitate the monitoring of nucleoside-, nucleotide-, and nucleic acid-based transformations at a nucleobase resolution in real time. The premise underlying our approach is the identification of minimal atomic/structural perturbations that endow the synthetic analogs with favorable photophysical features while maintaining native conformations and pairing. As illuminating probes, the photophysical parameters of such isomorphic nucleosides display sensitivity to microenvironmental factors. Responsive isomorphic analogs that function similarly to their native counterparts in biochemical contexts are defined as isofunctional.Early analogs included pyrimidines substituted with five-membered aromatic heterocycles at their 5 position and have been used to assess the polarity of the major groove in duplexes. Polarized quinazolines have proven useful in assembling FRET pairs with established fluorophores and have been used to study RNA-protein and RNA-small-molecule binding. Completing a fluorescent ribonucleoside alphabet, composed of visibly emissive purine (A, G) and pyrimidine (U, C) analogs, all derived from thieno[3,4-]pyrimidine as the heterocyclic nucleus, was a major breakthrough. To further augment functionality, a second-generation emissive RNA alphabet based on an isothiazolo[4,3-]pyrimidine core (A, G, U, and C) was fabricated. This single-atom "mutagenesis" restored the basic/coordinating nitrogen corresponding to N7 in the purine skeleton and elevated biological recognition.The isomorphic emissive nucleosides and nucleotides, particularly the purine analogs, serve as substrates for diverse enzymes. Beyond polymerases, we have challenged the emissive analogs with metabolic and catabolic enzymes, opening optical windows into the biochemistry of nucleosides and nucleotides as metabolites as well as coenzymes and second messengers. Real-time fluorescence-based assays for adenosine deaminase, guanine deaminase, and cytidine deaminase have been fabricated and used for inhibitor discovery. Emissive cofactors (e.g., SAM), coenzymes (e.g., NAD), and second messengers (e.g., c-di-GMP) have been enzymatically synthesized, using NTPs and native enzymes. Both their biosynthesis and their transformations can be fluorescently monitored in real time.Highly isomorphic and isofunctional emissive surrogates can therefore be fabricated and judiciously implemented. Beyond their utility, side-by-side comparison to established analogs, particularly to 2-aminopurine, the workhorse of nucleic acid biophysics over 5 decades, has proven prudent as they refined the scope and limitations of both the new analogs and their predecessors. Challenges, however, remain. Associated with such small heterocycles are relatively short emission wavelengths and limited brightness. Recent advances in multiphoton spectroscopy and further structural modifications have shown promise for overcoming such barriers.
Topics: Fluorescent Dyes; Nucleosides; RNA; DNA
PubMed: 38613490
DOI: 10.1021/acs.accounts.4c00042 -
ZNF432 stimulates PARylation and inhibits DNA resection to balance PARPi sensitivity and resistance.Nucleic Acids Research Nov 2023Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of...
Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.
Topics: Humans; DNA; DNA Damage; DNA Repair; Poly (ADP-Ribose) Polymerase-1; Poly Adenosine Diphosphate Ribose; Poly ADP Ribosylation
PubMed: 37823600
DOI: 10.1093/nar/gkad791 -
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 -
Briefings in Bioinformatics Jul 2023Nucleic acid-binding proteins are proteins that interact with DNA and RNA to regulate gene expression and transcriptional control. The pathogenesis of many human...
Nucleic acid-binding proteins are proteins that interact with DNA and RNA to regulate gene expression and transcriptional control. The pathogenesis of many human diseases is related to abnormal gene expression. Therefore, recognizing nucleic acid-binding proteins accurately and efficiently has important implications for disease research. To address this question, some scientists have proposed the method of using sequence information to identify nucleic acid-binding proteins. However, different types of nucleic acid-binding proteins have different subfunctions, and these methods ignore their internal differences, so the performance of the predictor can be further improved. In this study, we proposed a new method, called iDRPro-SC, to predict the type of nucleic acid-binding proteins based on the sequence information. iDRPro-SC considers the internal differences of nucleic acid-binding proteins and combines their subfunctions to build a complete dataset. Additionally, we used an ensemble learning to characterize and predict nucleic acid-binding proteins. The results of the test dataset showed that iDRPro-SC achieved the best prediction performance and was superior to the other existing nucleic acid-binding protein prediction methods. We have established a web server that can be accessed online: http://bliulab.net/iDRPro-SC.
Topics: Humans; DNA-Binding Proteins; RNA-Binding Proteins; DNA; Algorithms
PubMed: 37405873
DOI: 10.1093/bib/bbad251 -
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