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1.
Mol Cell ; 80(2): 177-180, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33065018

RESUMO

In this issue of Molecular Cell, Kim et al., 2020 report that PCAF is a fork-associated histone acetyltransferase (HAT) that regulates the stability of stalled forks and the response to PARP inhibition in BRCA1/2-deficient cells.


Assuntos
Replicação do DNA , Histonas , Acetilação , Endonucleases
2.
Mol Cell ; 80(1): 6-8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007257

RESUMO

Kapadia et al. (2020) use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.


Assuntos
DNA Polimerase Dirigida por DNA , Células Eucarióticas , DNA , Replicação do DNA
3.
Mol Cell ; 79(6): 1037-1050.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32882183

RESUMO

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.


Assuntos
DNA/genética , RNA/genética , Saccharomyces cerevisiae/genética , Adolescente , Adulto , DNA/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Instabilidade Genômica/genética , Humanos , Pessoa de Meia-Idade , RNA/ultraestrutura , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Adulto Jovem
4.
Nat Commun ; 11(1): 4534, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913330

RESUMO

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proliferação de Células/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Estruturas R-Loop/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina , Dano ao DNA , Instabilidade Genômica , Humanos , Neoplasias/patologia , Transcrição Genética
5.
Mol Cell ; 80(1): 114-126.e8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916094

RESUMO

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Tempo
6.
Adv Exp Med Biol ; 1267: 59-80, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894477

RESUMO

The internal spatial organization of prokaryotic organisms, including Escherichia coli, is essential for the proper functioning of processes such as cell division. One source of this organization in E. coli is the nucleoid, which causes the exclusion of macromolecules - e.g. protein aggregates and the chemotaxis network - from midcell. Similarly, following DNA replication, the nucleoid(s) assist in placing the Z-ring at midcell. These processes need to be efficient in optimal conditions and robust to suboptimal conditions. After reviewing recent findings on these topics, we make use of past data to study the efficiency of the spatial constraining of Z-rings, chemotaxis networks, and protein aggregates, as a function of the nucleoid(s) morphology. Also, we compare the robustness of these processes to nonoptimal temperatures. We show that Z-rings, Tsr clusters, and protein aggregates have temperature-dependent spatial distributions along the major cell axis that are consistent with the nucleoid(s) morphology and the volume-exclusion phenomenon. Surprisingly, the consequences of the changes in nucleoid size with temperature are most visible in the kurtosis of these spatial distributions, in that it has a statistically significant linear correlation with the mean nucleoid length and, in the case of Z-rings, with the distance between nucleoids prior to cell division. Interestingly, we also find a negative, statistically significant linear correlation between the efficiency of these processes at the optimal condition and their robustness to suboptimal conditions, suggesting a trade-off between these traits.


Assuntos
Escherichia coli/citologia , Escherichia coli/metabolismo , Organelas/metabolismo , Divisão Celular , Replicação do DNA
7.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 42(4): 485-490, 2020 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895100

RESUMO

Objective To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Methods Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Results Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(t=6.397,P=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(t=8.241,P=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(t=2.916,P=0.0434;t=3.452,P=0.0260). Conclusion In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.


Assuntos
Replicação do DNA , Dano ao DNA , Papillomavirus Humano 16 , Mitose , Proteínas Oncogênicas Virais
8.
Nat Commun ; 11(1): 4845, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973148

RESUMO

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of ß-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases.


Assuntos
Replicação do DNA/fisiologia , Infecções por Herpesviridae/metabolismo , Herpesviridae/metabolismo , Proteínas Quinases/metabolismo , Animais , Ciclina A/genética , Ciclina A/metabolismo , Citomegalovirus/genética , DNA/metabolismo , Células HEK293 , Herpesviridae/enzimologia , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Células NIH 3T3 , Sinais de Localização Nuclear/metabolismo , Fosforilação , Mapas de Interação de Proteínas , Proteínas Virais/genética , Proteínas Virais/metabolismo
9.
Mol Cell ; 79(5): 705-707, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32888435

RESUMO

In this issue of Molecular Cell, Benslimane et al. (2020) perform a CRISPR-Cas9 chemogenomic screen, identifying a network of DNA replication and genome integrity genes with the nutraceutical compound Resveratrol and its analog Pterostilbene, linking these compounds to the induction of DNA replication stress in mammalian cells.


Assuntos
Replicação do DNA , Resveratrol , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos
10.
Nat Commun ; 11(1): 4894, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994400

RESUMO

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.


Assuntos
Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Replicação do DNA , Modelos Animais de Doenças , Encefalite por Herpes Simples/patologia , Feminino , Genes Virais/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Camundongos , Biossíntese de Proteínas , Proteômica/métodos , Células Vero , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral
11.
Nat Commun ; 11(1): 4826, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958757

RESUMO

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.


Assuntos
DNA/biossíntese , DNA/química , Origem de Replicação/genética , Animais , Composição de Bases , Sequência de Bases , Carcinogênese , Diferenciação Celular , Células Cultivadas , Replicação do DNA/genética , Genoma Humano/genética , Heterocromatina/genética , Humanos , Camundongos , Motivos de Nucleotídeos , Transcrição Genética
12.
Sci Data ; 7(1): 314, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963239

RESUMO

Establishing consensus around the transcriptional interface between coronavirus (CoV) infection and human cellular signaling pathways can catalyze the development of novel anti-CoV therapeutics. Here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in MERS-CoV (MERS), SARS-CoV-1 (SARS1) and SARS-CoV-2 (SARS2)-infected cells. Validating the CoV consensomes, we show that high confidence transcriptional targets (HCTs) of MERS, SARS1 and SARS2 infection intersect with HCTs of signaling pathway nodes with known roles in CoV infection. Among a series of novel use cases, we gather evidence for hypotheses that SARS2 infection efficiently represses E2F family HCTs encoding key drivers of DNA replication and the cell cycle; that progesterone receptor signaling antagonizes SARS2-induced inflammatory signaling in the airway epithelium; and that SARS2 HCTs are enriched for genes involved in epithelial to mesenchymal transition. The CoV infection consensomes and HCT intersection analyses are freely accessible through the Signaling Pathways Project knowledgebase, and as Cytoscape-style networks in the Network Data Exchange repository.


Assuntos
Infecções por Coronavirus/genética , Transição Epitelial-Mesenquimal/genética , Pneumonia Viral/genética , Transcriptoma , Betacoronavirus , Ciclo Celular , Consenso , Replicação do DNA , Conjuntos de Dados como Assunto , Expressão Gênica , Humanos , Fatores Reguladores de Interferon/genética , Coronavírus da Síndrome Respiratória do Oriente Médio , Pandemias , Receptores de Progesterona , Vírus da SARS , Transdução de Sinais
13.
PLoS Genet ; 16(8): e1008987, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853297

RESUMO

Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This gene strand bias arises primarily due to negative selection against deleterious consequences of head-on replication-transcription conflict. However, many genes remain head-on. Can head-on orientation provide some benefit? We combine insights from both mechanistic and evolutionary studies, review published work, and analyze gene expression data to evaluate an emerging model that head-on genes are temporal targets for adaptive mutagenesis during stress. We highlight the alternative explanation that genes in the head-on orientation may simply be the result of genomic inversions and relaxed selection acting on nonessential genes. We seek to clarify how the mechanisms of replication-transcription conflict, in concert with other mutagenic mechanisms, balanced by natural selection, have shaped bacterial genome evolution.


Assuntos
Replicação do DNA/genética , Evolução Molecular , Seleção Genética/genética , Transcrição Genética , Bactérias/genética , Genoma Bacteriano/genética
14.
Nat Commun ; 11(1): 3940, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769985

RESUMO

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo
15.
Mol Cell ; 79(5): 846-856.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755594

RESUMO

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.


Assuntos
Proliferação de Células/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resveratrol/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Resistência a Medicamentos/genética , Humanos , Hidroxiureia/farmacologia , Células Jurkat , Nucleotídeos/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sirtuína 1/metabolismo , Estilbenos/farmacologia
16.
Nat Commun ; 11(1): 3796, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732900

RESUMO

The ter region of the bacterial chromosome, where replication terminates, is the last to be segregated before cell division in Escherichia coli. Delayed segregation is controlled by the MatP protein, which binds to specific sites (matS) within ter, and interacts with other proteins such as ZapB. Here, we investigate the role of MatP by combining short-time mobility analyses of the ter locus with biochemical approaches. We find that ter mobility is similar to that of a non ter locus, except when sister ter loci are paired after replication. This effect depends on MatP, the persistence of catenanes, and ZapB. We characterise MatP/DNA complexes and conclude that MatP binds DNA as a tetramer, but bridging matS sites in a DNA-rich environment remains infrequent. We propose that tetramerisation of MatP links matS sites with ZapB and/or with non-specific DNA to promote optimal pairing of sister ter regions until cell division.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Divisão Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo
17.
Nat Commun ; 11(1): 3813, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732913

RESUMO

Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by the E. coli nucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes.


Assuntos
Bacteriófago lambda/genética , Replicação do DNA/genética , Escherichia coli/virologia , Montagem de Vírus/genética , Bacteriófago lambda/crescimento & desenvolvimento , DNA Viral/biossíntese , DNA Viral/genética , Escherichia coli/genética , Lisogenia/genética , Transcrição Genética/genética
18.
Phys Rev Lett ; 125(4): 048104, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32794805

RESUMO

The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.


Assuntos
Ecossistema , RNA/química , RNA/genética , Catálise , DNA/química , DNA/genética , DNA/metabolismo , Replicação do DNA , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Planeta Terra , Evolução Molecular , Temperatura Alta , Biossíntese de Proteínas/genética , RNA/metabolismo
19.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826907

RESUMO

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Assuntos
Proteínas de Bactérias/química , DNA Primase/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
20.
Nat Commun ; 11(1): 4083, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796829

RESUMO

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.


Assuntos
Proteínas de Ciclo Celular/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Estruturas R-Loop/efeitos dos fármacos , Fatores de Transcrição/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HeLa , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Fase S , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
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