RESUMO
DNA replication and transcription occur in all living cells across all domains of life. Both essential processes occur simultaneously on the same template, leading to conflicts between the macromolecular machines that perform these functions. Numerous studies over the past few decades demonstrate that this is an inevitable problem in both prokaryotic and eukaryotic cells. We have learned that conflicts lead to replication fork reversal, breaks in the DNA, R-loop formation, topological stress, and mutagenesis and can ultimately impact evolution. Recent studies have also provided insight into the various mechanisms that mitigate, resolve, and allow tolerance of conflicts and how conflicts result in pathological consequences across divergent species. In this review, we summarize our current knowledge regarding the outcomes of the encounters between replication and transcription machineries and explore how these clashes are dealt with across species.
Assuntos
Replicação do DNA , Transcrição Gênica , Humanos , Animais , Cromossomos/metabolismo , Cromossomos/genética , Cromossomos/química , Estruturas R-Loop , DNA/metabolismo , DNA/genética , DNA/químicaRESUMO
DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.
Assuntos
Reparo do DNA , Poli(ADP-Ribose) Polimerase-1 , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , HumanosRESUMO
Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.
Assuntos
Mecanotransdução Celular , Imagem Individual de Molécula , Animais , Humanos , Camundongos , Fenômenos Biomecânicos , Adesão Celular , DNA/química , DNA/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Microscopia de Força Atômica/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Sobrevivência Celular , Pareamento de Bases , CalibragemRESUMO
Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.
Assuntos
Proteínas de Ligação a DNA , Desenvolvimento Embrionário , Fatores de Transcrição , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Mesoderma/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Animais , Camundongos , Extremidades/crescimento & desenvolvimentoRESUMO
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , DNA , Edição de Genes , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/metabolismo , DNA/genética , Edição de Genes/métodos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HEK293 , Domínios Proteicos , Genoma Humano , Modelos Moleculares , Estrutura Terciária de Proteína , Conformação de Ácido Nucleico , Biocatálise , Magnésio/química , Magnésio/metabolismoRESUMO
DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteína 9 Associada à CRISPR/genética , Genoma , DNA/genética , DNA/metabolismoRESUMO
In this autobiographical article, I reflect on my Swedish background. Then I discuss endogenous DNA alterations and the base excision repair pathway and alternative repair strategies for some unusual DNA lesions. Endogenous DNA damage, such as loss of purine bases and cytosine deamination, is proposed as a major source of cancer-causing mutations.
Assuntos
DNA Glicosilases , Reparo do DNA , Dano ao DNA , DNA/genética , DNA/metabolismo , DNA Glicosilases/metabolismoRESUMO
SMC (structural maintenance of chromosomes) protein complexes are an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. Here, we describe the roles of SMCs in chromosome biology and particularly review in vitro single-molecule studies that have recently advanced our understanding of SMC proteins. We describe the mechanistic biophysical aspects of loop extrusion that govern genome organization and its consequences.
Assuntos
Proteínas Cromossômicas não Histona , Complexos Multiproteicos , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/química , Cromossomos/genética , Cromossomos/metabolismo , DNA/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors.
Assuntos
Síndrome de Cockayne , Humanos , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Transcrição Gênica , Reparo do DNA , Dano ao DNA , DNA/genética , DNA/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.
Assuntos
Imunidade Inata , Nucleotidiltransferases , Humanos , Animais , Nucleotidiltransferases/metabolismo , Imunidade Inata/genética , Transdução de Sinais/genética , DNA/metabolismo , Receptores de Reconhecimento de PadrãoRESUMO
ADP-ribosylation is a ubiquitous modification of biomolecules, including proteins and nucleic acids, that regulates various cellular functions in all kingdoms of life. The recent emergence of new technologies to study ADP-ribosylation has reshaped our understanding of the molecular mechanisms that govern the establishment, removal, and recognition of this modification, as well as its impact on cellular and organismal function. These advances have also revealed the intricate involvement of ADP-ribosylation in human physiology and pathology and the enormous potential that their manipulation holds for therapy. In this review, we present the state-of-the-art findings covering the work in structural biology, biochemistry, cell biology, and clinical aspects of ADP-ribosylation.
Assuntos
ADP-Ribosilação , Humanos , Proteínas/metabolismo , DNA/metabolismo , RNA/metabolismo , Animais , Transdução de Sinais , Processamento de Proteína Pós-Traducional , ADP Ribose Transferases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Gene regulation arises out of dynamic competition between nucleosomes, transcription factors, and other chromatin proteins for the opportunity to bind genomic DNA. The timescales of nucleosome assembly and binding of factors to DNA determine the outcomes of this competition at any given locus. Here, we review how these properties of chromatin proteins and the interplay between the dynamics of different factors are critical for gene regulation. We discuss how molecular structures of large chromatin-associated complexes, kinetic measurements, and high resolution mapping of protein-DNA complexes in vivo set the boundary conditions for chromatin dynamics, leading to models of how the steady state behaviors of regulatory elements arise.
Assuntos
Cromatina , Nucleossomos , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Nucleossomos/genética , Fatores de Transcrição/genéticaRESUMO
Our current view of how DNA-based genomes are efficiently and accurately replicated continues to evolve as new details emerge on the presence of ribonucleotides in DNA. Ribonucleotides are incorporated during eukaryotic DNA replication at rates that make them the most common noncanonical nucleotide placed into the nuclear genome, they are efficiently repaired, and their removal impacts genome integrity. This review focuses on three aspects of this subject: the incorporation of ribonucleotides into the eukaryotic nuclear genome during replication by B-family DNA replicases, how these ribonucleotides are removed, and the consequences of their presence or removal for genome stability and disease.
Assuntos
Replicação do DNA , Instabilidade Genômica , Ribonucleotídeos , DNA/genética , DNA/metabolismo , Reparo do DNA , Eucariotos/genética , Eucariotos/metabolismo , Nucleotidiltransferases/genética , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismoRESUMO
Covalent DNA-protein crosslinks (DPCs) are pervasive DNA lesions that interfere with essential chromatin processes such as transcription or replication. This review strives to provide an overview of the sources and principles of cellular DPC formation. DPCs are caused by endogenous reactive metabolites and various chemotherapeutic agents. However, in certain conditions DPCs also arise physiologically in cells. We discuss the cellular mechanisms resolving these threats to genomic integrity. Detection and repair of DPCs require not only the action of canonical DNA repair pathways but also the activity of specialized proteolytic enzymes-including proteases of the SPRTN/Wss1 family-to degrade the crosslinked protein. Loss of DPC repair capacity has dramatic consequences, ranging from genome instability in yeast and worms to cancer predisposition and premature aging in mice and humans.
Assuntos
Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Animais , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Camundongos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.
Assuntos
Proteínas Argonautas , Células Procarióticas/fisiologia , Proteínas Argonautas/metabolismo , DNA/metabolismo , Células Procarióticas/citologia , Células Procarióticas/metabolismo , RNA Guia de CinetoplastídeosRESUMO
Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.
Assuntos
Sistemas de Liberação de Medicamentos , Engenharia Genética , Proteínas/uso terapêutico , Vírion/genética , Animais , Sequência de Bases , Cegueira/genética , Cegueira/terapia , Encéfalo/metabolismo , DNA/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Edição de Genes , Células HEK293 , Humanos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Epitélio Pigmentado da Retina/patologia , Retroviridae , Vírion/ultraestrutura , Visão OcularRESUMO
Endogenous cytoplasmic DNA (cytoDNA) species are emerging as key mediators of inflammation in diverse physiological and pathological contexts. Although the role of endogenous cytoDNA in innate immune activation is well established, the cytoDNA species themselves are often poorly characterized and difficult to distinguish, and their mechanisms of formation, scope of function and contribution to disease are incompletely understood. Here, we summarize current knowledge in this rapidly progressing field with emphases on similarities and differences between distinct cytoDNAs, their underlying molecular mechanisms of formation and function, interactions between cytoDNA pathways, and therapeutic opportunities in the treatment of age-associated diseases.
Assuntos
Envelhecimento/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , Doença , Animais , Humanos , Micronúcleo Germinativo/metabolismo , Retroelementos/genéticaRESUMO
While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.
Assuntos
Edição de Genes , Sistemas CRISPR-Cas/genética , Linhagem Celular , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Feminino , Genes Dominantes , Genoma Humano , Humanos , Masculino , Modelos Biológicos , Proteína 1 Homóloga a MutL/genética , Mutação/genética , RNA/metabolismo , Reprodutibilidade dos TestesRESUMO
Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Conformação de Ácido Nucleico , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformação Proteica , CoesinasRESUMO
RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.