Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.453
Filtrar
1.
Proc Natl Acad Sci U S A ; 117(32): 19475-19486, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709741

RESUMO

The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4+ T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4+ T cells, plasmid challenge or HSV-1ΔUL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , DNA Viral/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Especificidade da Espécie , Replicação Viral
2.
Proc Natl Acad Sci U S A ; 117(26): 14936-14947, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541055

RESUMO

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.


Assuntos
Proteínas Arqueais/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Sulfolobus acidocaldarius/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/genética , Microscopia de Força Atômica , Ligação Proteica , Sulfolobus acidocaldarius/química , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/metabolismo
3.
Nat Commun ; 11(1): 3088, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555206

RESUMO

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.


Assuntos
Quebras de DNA de Cadeia Dupla , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Western Blotting , Linhagem Celular Tumoral , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Exodesoxirribonucleases/genética , Imunofluorescência , Recombinação Homóloga/genética , Humanos , RecQ Helicases/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
4.
Am J Med Sci ; 360(3): 287-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32387117

RESUMO

Immunoglobulin A (IgA) nephropathy is one of the most common glomerulonephritis characterized by the deposition of IgA in glomerular mesangium. Karyomegalic interstitial nephritis (KIN) is a rare interstitial nephritis with potential hereditary factors. IgA nephropathy concomitant with KIN has not yet been reported. Herein, we describe the clinical course, ultrasonic images and gastrointestinal endoscopy findings of a 28-year-old-male patient with IgA nephropathy with KIN. The pathologic examination of the renal biopsy specimen demonstrated mild mesangial proliferative IgA nephropathy with KIN. Molecular genetic testing detected an abnormality in FAN1 gene. The heterozygous mutation was present on chromosome 15q13.3. However, IgA nephropathy with KIN is a rare disorder, and its pathogenesis is yet to be clarified.


Assuntos
Glomerulonefrite por IGA/patologia , Nefrite Intersticial/genética , Nefrite Intersticial/patologia , Adulto , Cromossomos Humanos Par 15/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Mutação da Fase de Leitura , Mesângio Glomerular , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/fisiopatologia , Heterozigoto , Humanos , Testes de Função Renal , Masculino , Enzimas Multifuncionais/genética , Mutação de Sentido Incorreto , Nefrite Intersticial/fisiopatologia
5.
Science ; 368(6488)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32299917

RESUMO

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. We explain this association by elucidating a mutational cascade that is triggered by a single cell division error-chromosome bridge formation-that rapidly increases genomic complexity. We show that actomyosin forces are required for initial bridge breakage. Chromothripsis accumulates, beginning with aberrant interphase replication of bridge DNA. A subsequent burst of DNA replication in the next mitosis generates extensive DNA damage. During this second cell division, broken bridge chromosomes frequently missegregate and form micronuclei, promoting additional chromothripsis. We propose that iterations of this mutational cascade generate the continuing evolution and subclonal heterogeneity characteristic of many human cancers.


Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Quebra Cromossômica , Dano ao DNA/genética , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Actomiosina/metabolismo , Linhagem Celular Tumoral , Exodesoxirribonucleases/genética , Dosagem de Genes , Genoma Humano , Humanos , Fenômenos Mecânicos , Mutagênese , Mutação , Fosfoproteínas/genética , Análise de Célula Única
6.
PLoS Genet ; 16(4): e1008733, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32287268

RESUMO

In budding yeast, Cdc13, Stn1, and Ten1 form the telomere-binding heterotrimer CST complex. Here we investigate the role of Cdc13/CST in maintaining genome stability by using a Chr VII disome system that can generate recombinants, chromosome loss, and enigmatic unstable chromosomes. In cells expressing a temperature sensitive CDC13 allele, cdc13F684S, unstable chromosomes frequently arise from problems in or near a telomere. We found that, when Cdc13 is defective, passage through S phase causes Exo1-dependent ssDNA and unstable chromosomes that are then the source for additional chromosome instability events (e.g. recombinants, chromosome truncations, dicentrics, and/or chromosome loss). We observed that genome instability arises from a defect in Cdc13's function during DNA replication, not Cdc13's putative post-replication telomere capping function. The molecular nature of the initial unstable chromosomes formed by a Cdc13-defect involves ssDNA and does not involve homologous recombination nor non-homologous end joining; we speculate the original unstable chromosome may be a one-ended double strand break. This system defines a link between Cdc13's function during DNA replication and genome stability in the form of unstable chromosomes, that then progress to form other chromosome changes.


Assuntos
Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Recombinação Genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética
7.
Nucleic Acids Res ; 48(9): 4960-4975, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232411

RESUMO

G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.


Assuntos
Enzimas Reparadoras do DNA/fisiologia , Replicação do DNA , Exodesoxirribonucleases/fisiologia , Quadruplex G , Telômero/química , Aminoquinolinas/farmacologia , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Quadruplex G/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidade , Ácidos Picolínicos/farmacologia , Prognóstico
8.
Nucleic Acids Res ; 48(6): 3053-3070, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32020204

RESUMO

The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.


Assuntos
Proteínas 14-3-3/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Exodesoxirribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Genoma Fúngico/genética , Instabilidade Genômica/genética , Fosforilação/genética , Processamento de Proteína Pós-Traducional/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/genética
9.
Nat Commun ; 11(1): 857, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051414

RESUMO

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm-/- spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.


Assuntos
Endodesoxirribonucleases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Espermatócitos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Proteína Homóloga a MRE11/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
10.
Stroke ; 51(1): 300-307, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31805844

RESUMO

Background and Purpose- Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an autosomal dominant small vessel disease caused by C-terminal frameshift mutations in the TREX1 gene that encodes the major mammalian 3' to 5' DNA exonuclease. RVCL-S is characterized by vasculopathy, especially in densely vascularized organs, progressive retinopathy, cerebral microvascular disease, white matter lesions, and migraine, but the underlying mechanisms are unknown. Methods- Homozygous transgenic RVCL-S knock-in mice expressing a truncated Trex1 (three prime repair exonuclease 1) protein (similar to what is seen in patients) and wild-type littermates, of various age groups, were subjected to (1) a survival analysis, (2) in vivo postocclusive reactive hyperemia and ex vivo Mulvany myograph studies to characterize the microvascular and macrovascular reactivity, and (3) experimental stroke after transient middle cerebral artery occlusion with neurological deficit assessment. Results- The mutant mice show increased mortality starting at midlife (P=0.03 with hazard ratio, 3.14 [95% CI, 1.05-9.39]). The mutants also show a vascular phenotype as evidenced by attenuated postocclusive reactive hyperemia responses (across all age groups; F[1, 65]=5.7, P=0.02) and lower acetylcholine-induced relaxations in aortae (in 20- to 24-month-old mice; RVCL-S knock-in: Emax: 37±8% versus WT: Emax: 65±6%, P=0.01). A vascular phenotype is also suggested by the increased infarct volume seen in 12- to 14-month-old mutant mice at 24 hours after infarct onset (RVCL-S knock-in: 75.4±2.7 mm3 versus WT: 52.9±5.6 mm3, P=0.01). Conclusions- Homozygous RVCL-S knock-in mice show increased mortality, signs of abnormal vascular function, and increased sensitivity to experimental stroke and can be instrumental to investigate the pathology seen in patients with RVCL-S.


Assuntos
Exodesoxirribonucleases , Leucoencefalopatias , Fosfoproteínas , Doenças Retinianas , Doenças Vasculares , Animais , Modelos Animais de Doenças , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Técnicas de Introdução de Genes , Humanos , Leucoencefalopatias/enzimologia , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Camundongos , Camundongos Mutantes , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Doenças Retinianas/enzimologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Doenças Vasculares/enzimologia , Doenças Vasculares/genética , Doenças Vasculares/patologia
11.
J Surg Oncol ; 121(5): 906-916, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31650563

RESUMO

BACKGROUND AND OBJECTIVES: DNA repair is a new and important pathway that explains colorectal carcinogenesis. This study will evaluate the prognostic value of molecular modulation of double-strand break repair (XRCC2 and XRCC5); DNA damage tolerance/translesion synthesis (POLH, POLK, and POLQ), and interstrand crosslink repair (DCLRE1A) in sporadic colorectal cancer (CRC). METHODS: Tumor specimens and matched healthy mucosal tissues from 47 patients with CRC who underwent surgery were assessed for gene expression of XRCC2, XRCC5, POLH, POLK, POLQ, and DCLRE1A; protein expression of Polk, Ku80, p53, Ki67, and mismatch repair MLH1 and MSH2 components; CpG island promoter methylation of XRCC5, POLH, POLK, POLQ, and DCLRE1A was performed. RESULTS: Neoplastic tissues exhibited induction of POLK (P < .001) and DCLRE1A (P < .001) expression and low expression of POLH (P < .001) and POLQ (P < .001) in comparison to healthy paired mucosa. Low expression of POLH was associated with mucinous histology and T1-T2 tumors (P = .038); low tumor expression of POLK was associated with distant metastases (P = .042). CRC harboring POLK promoter methylation exhibited better disease-free survival (DFS) (P = .005). CONCLUSIONS: This study demonstrated that low expression or unmethylated POLH and POLK were related to worse biological behavior tumors. However, POLK methylation was associated with better DFS. POLK and POLH are potential prognostic biomarkers in CRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Ilhas de CpG , Dano ao DNA , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Intervalo Livre de Doença , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Expressão Gênica , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Masculino , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Metástase Neoplásica/genética , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Nucleic Acids Res ; 48(3): 1271-1284, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31828313

RESUMO

The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.


Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Reparo de DNA por Recombinação/genética , Proteínas de Schizosaccharomyces pombe/genética , Telômero/genética , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Exodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico/genética , Instabilidade Genômica/genética , Perda de Heterozigosidade/genética , Rad51 Recombinase/genética , Schizosaccharomyces/genética
13.
Mol Cell ; 77(2): 395-410.e3, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31759824

RESUMO

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.


Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Replicação do DNA/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Nucleossomos/genética , RecQ Helicases/genética , Saccharomyces cerevisiae/genética
14.
Viruses ; 12(1)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861565

RESUMO

Human T-lymphotropic virus type 1 (HTLV-1) deregulates the immune system and cell cycle, resulting in loss of immune tolerance and disease, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Three prime repair exonuclease 1 (TREX1) maintains innate immune tolerance of the host and host-cell permissiveness to retroviral infections. TREX1 polymorphisms may influence the course of infection and autoimmune manifestations. The influence of TREX1 531C/T polymorphism was investigated in HTLV-1 infection and development of symptoms among 151 persons infected with HTLV-1 (32 HAM/TSP, 19 rheumatologic manifestations, two dermatitis, five more than one diagnosis, two probable HAM/TSP, and 91 asymptomatic individuals) and 100 uninfected persons in the control group. Polymorphism genotyping and proviral load quantification were performed by real-time polymerase chain reaction (PCR) and antinuclear antibodies (ANAs) were screened by an indirect immunofluorescence assay. No statistically significant difference was found in polymorphism genotype and allele frequencies between the infected and control groups. HAM/TSP patients showed higher frequency of TT genotype than asymptomatic persons (p = 0.0339). Proviral load was significantly higher among individuals with CT/TT genotypes and CC genotype carriers had lower proviral load and higher levels of proinflammatory cytokines. ANAs were present only in the HAM/TSP group. TREX1 531C>T polymorphism seems to be associated with TREX-1 regulation and HTLV-1 infection.


Assuntos
Exodesoxirribonucleases/genética , Predisposição Genética para Doença , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Carga Viral , Alelos , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino
15.
Genes (Basel) ; 10(11)2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683605

RESUMO

Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.


Assuntos
Adaptação Fisiológica , Sistemas CRISPR-Cas , Reparo do DNA , Escherichia coli/genética , Instabilidade Genômica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Genoma Bacteriano
16.
Mol Biotechnol ; 61(12): 938-944, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31641996

RESUMO

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonucleases/genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Temperatura Alta , Mutação , Temperatura
17.
Mol Cell ; 76(5): 699-711.e6, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31542296

RESUMO

Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from ∼3-5 kb/h up to ∼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Cinética , Mutação , Domínios Proteicos , Transporte Proteico , Proteína Rad52 de Recombinação e Reparo de DNA/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
18.
Mol Cell ; 75(3): 605-619.e6, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31255466

RESUMO

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Exodesoxirribonucleases/genética , RecQ Helicases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Instabilidade Genômica/genética , Células HeLa , Humanos , Neoplasias/genética , Mutações Sintéticas Letais/genética
19.
Mol Cell Probes ; 46: 101423, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31323319

RESUMO

Polydeoxyadenosine (poly (dA)) has been extensively applied for detecting many drug molecules. Herein, we developed a sensitive method for detecting coralyne and heparin using a modified DNA probe with poly (dA) at one end. In the absence of coralyne, the DNA probe was digested by the Exonuclease I (Exo I), and therefore the SYBR Green I (SG I) emitted an extremely low fluorescent signal. While coralyne specifically binding to poly (dA) with strong propensity could remarkably restrain the disintegration of the DNA probe, through which as a template the second strand of DNA sequence was formed with the introduction of DNA polymerase. Therefore, the fluorescent signal of SG I was intensified to quantify coralyne. Based on this method, heparin can be determined due to its strong affinity towards coralyne. This method showed a linear range from 2 to 500 nM for coralyne with a low detection limit of 0.98 nM, and the linear range of heparin was from 1 to 100 nM when 1.25 nm was the detection limit. The proposed method was also implemented successfully in biological samples and showed a potential application for screening potential therapeutic molecules.


Assuntos
Alcaloides de Berberina/isolamento & purificação , Técnicas Biossensoriais , Exodesoxirribonucleases/genética , Heparina/isolamento & purificação , Alcaloides de Berberina/química , DNA/química , Sondas de DNA/química , Sondas de DNA/genética , Desoxiadenosinas/química , Desoxiadenosinas/genética , Exodesoxirribonucleases/química , Heparina/química , Heparina/genética , Humanos , Limite de Detecção , Compostos Orgânicos/química
20.
Nucleic Acids Res ; 47(16): 8521-8536, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31251806

RESUMO

DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5' overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , DNA Polimerase I/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Dano ao DNA , DNA Polimerase I/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA