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
Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.
Assuntos
Duplicação Gênica , Edição de Genes , Genoma Humano , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , DNA/genética , Animais , Células-Tronco Embrionárias/metabolismo , Cromossomos Humanos/genéticaRESUMO
Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição SOXB1 , Super Intensificadores , Transcrição Gênica , DNA/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição SOXB1/genética , Animais , Camundongos , Células-Tronco Embrionárias/metabolismo , Microscopia/métodosRESUMO
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
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
Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.
Assuntos
Citidina Desaminase , Hipermutação Somática de Imunoglobulina , Animais , Camundongos , Anticorpos/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , DNA de Cadeia Simples , Mutação , Evolução Molecular , Regiões Determinantes de Complementaridade/genética , Motivos de NucleotídeosRESUMO
Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.
Assuntos
Proteína de Replicação A , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Camundongos , DNA/genética , Reparo de Erro de Pareamento de DNA , Doença de Huntington/genética , Proteínas/genética , Ataxias Espinocerebelares/genética , Proteína de Replicação A/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
In the decade since the discovery of the innate immune cyclic GMP-AMP synthase (cGAS)-2'3'-cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway, its proper activation and dysregulation have been rapidly implicated in many aspects of human disease. Understanding the biochemical, cellular, and regulatory mechanisms of this pathway is critical to developing therapeutic strategies that either harness it to boost defense or inhibit it to prevent unwanted inflammation. In this review, we first discuss how the second messenger cGAMP is synthesized by cGAS in response to double-stranded DNA and cGAMP's subsequent activation of cell-type-dependent STING signaling cascades with differential physiological consequences. We then review how cGAMP as an immunotransmitter mediates tightly controlled cell-cell communication by being exported from producing cells and imported into responding cells via cell-type-specific transporters. Finally, we review mechanisms by which thecGAS-cGAMP-STING pathway responds to different sources of mislocalized double-stranded DNA in pathogen defense, cancer, and autoimmune diseases.
Assuntos
Proteínas de Membrana , Nucleotídeos Cíclicos , DNA/genética , Humanos , Imunidade Inata/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/genética , Nucleotidiltransferases/genéticaRESUMO
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
Spatially resolved transcriptomic technologies are promising tools to study complex biological processes such as mammalian embryogenesis. However, the imbalance between resolution, gene capture, and field of view of current methodologies precludes their systematic application to analyze relatively large and three-dimensional mid- and late-gestation embryos. Here, we combined DNA nanoball (DNB)-patterned arrays and in situ RNA capture to create spatial enhanced resolution omics-sequencing (Stereo-seq). We applied Stereo-seq to generate the mouse organogenesis spatiotemporal transcriptomic atlas (MOSTA), which maps with single-cell resolution and high sensitivity the kinetics and directionality of transcriptional variation during mouse organogenesis. We used this information to gain insight into the molecular basis of spatial cell heterogeneity and cell fate specification in developing tissues such as the dorsal midbrain. Our panoramic atlas will facilitate in-depth investigation of longstanding questions concerning normal and abnormal mammalian development.
Assuntos
Organogênese , Transcriptoma , Animais , DNA/genética , Embrião de Mamíferos , Feminino , Perfilação da Expressão Gênica/métodos , Mamíferos/genética , Camundongos , Organogênese/genética , Gravidez , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genéticaRESUMO
Cell-type- and condition-specific profiles of gene expression require coordination between protein-coding gene promoters and cis-regulatory sequences called enhancers. Enhancers can stimulate gene activity at great genomic distances from their targets, raising questions about how enhancers communicate with specific gene promoters and what molecular mechanisms underlie enhancer function. Characterization of enhancer loci has identified the molecular features of active enhancers that accompany the binding of transcription factors and local opening of chromatin. These characteristics include coactivator recruitment, histone modifications, and noncoding RNA transcription. However, it remains unclear which of these features functionally contribute to enhancer activity. Here, we discuss what is known about how enhancers regulate their target genes and how enhancers and promoters communicate. Further, we describe recent data demonstrating many similarities between enhancers and the gene promoters they control, and we highlight unanswered questions in the field, such as the potential roles of transcription at enhancers.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genoma , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Transcrição Gênica , Animais , Cromatina/química , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Células Eucarióticas/metabolismo , Loci Gênicos , Código das Histonas , Histonas/genética , Histonas/metabolismo , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Transcription in several organisms from certain bacteria to humans has been observed to be stochastic in nature: toggling between active and inactive states. Periods of active nascent RNA synthesis known as bursts represent individual gene activation events in which multiple polymerases are initiated. Therefore, bursting is the single locus illustration of both gene activation and repression. Although transcriptional bursting was originally observed decades ago, only recently have technological advances enabled the field to begin elucidating gene regulation at the single-locus level. In this review, we focus on how biochemical, genomic, and single-cell data describe the regulatory steps of transcriptional bursts.
Assuntos
Cromatina/química , DNA/genética , Regulação da Expressão Gênica , Genoma , RNA Polimerase II/genética , RNA Mensageiro/genética , Transcrição Gênica , Animais , Cromatina/metabolismo , DNA/metabolismo , Células Eucarióticas/metabolismo , Loci Gênicos , Histonas/genética , Histonas/metabolismo , Humanos , Técnicas de Sonda Molecular , Sondas Moleculares/química , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Análise de Célula Única/métodos , Processos EstocásticosRESUMO
DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Animais , Coenzimas/química , Coenzimas/metabolismo , Ilhas de CpG , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Embrião de Mamíferos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Oócitos/citologia , Oócitos/enzimologia , Oócitos/crescimento & desenvolvimento , Transdução de Sinais , Espermatozoides/citologia , Espermatozoides/enzimologia , Espermatozoides/crescimento & desenvolvimentoRESUMO
DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.
Assuntos
DNA/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Genes Sintéticos , Engenharia Genética/métodos , Genoma , Sistemas CRISPR-Cas , DNA/química , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Poliovirus/genética , Poliovirus/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esferoplastos/genética , Esferoplastos/metabolismoRESUMO
Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks (DSBs), which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA DSBs, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression, and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA DSBs, in the budding yeast model system and in mammals.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Reparo do DNA por Junção de Extremidades , DNA/genética , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Humanos , Modelos Moleculares , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.