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1.
Front Physiol ; 12: 751374, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34690818

RESUMO

Physical activity (PA) and nutrition are the essential components of a healthy lifestyle, as they can influence energy balance, promote functional ability of various systems and improve immunity. Infections and their associated symptoms are the common and frequent challenges to human health that are causing severe economic and social consequences around the world. During aging, human immune system undergoes dramatic aging-related changes/dysfunctions known as immunosenescence. Clinically, immunosenescence refers to the gradual deterioration of immune system that increases exposure to infections, and reduces vaccine efficacy. Such phenomenon is linked to impaired immune responses that lead to dysfunction of multiple organs, while lack of physical activity, progressive loss of muscle mass, and concomitant decline in muscle strength facilitate immunosenescence and inflammation. In the present review, we have discussed the role of nutrition and PA, which can boost the immune system alone and synergistically. Evidence suggests that long-term PA is beneficial in improving immune system and preventing various infections. We have further discussed several nutritional strategies for improving the immune system. Unfortunately, the available evidence shows conflicting results. In terms of interaction with food intake, PA does not tend to increase energy intake during a short time course. However, overcoming nutritional deficiencies appears to be the most practical recommendation. Through the balanced nutritious diet intake one can fulfill the bodily requirement of optimal nutrition that significantly impacts the immune system. Supplementation of a single nutrient as food is generally not advisable. Rather incorporating various fruits and vegetables, whole grains, proteins and probiotics may ensure adequate nutrient intake. Therefore, multi-nutrient supplements may benefit people having deficiency in spite of sufficient diet. Along with PA, supplementation of probiotics, bovine colostrum, plant-derived products and functional foods may provide additional benefits in improving the immune system.

2.
Mol Cell ; 81(19): 4008-4025.e7, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34508659

RESUMO

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

3.
Nat Commun ; 12(1): 3937, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168151

RESUMO

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.


Assuntos
Replicação do DNA , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sobrevivência Celular , Dano ao DNA , Instabilidade Genômica , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Transporte de RNA
4.
J Am Soc Nephrol ; 32(6): 1339-1354, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33727367

RESUMO

BACKGROUND: MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression. METHODS: Selective ablation of Dicer in AQP2-expressing cells (DicerAQP2Cre+ mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice. RESULTS: The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in DicerAQP2Cre+ mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in DicerAQP2Cre+ mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409-3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the Aqp2 gene promoter in DicerAQP2Cre+ mice, resulting in decreased RNA Pol II association. CONCLUSIONS: Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.


Assuntos
Aquaporina 2/genética , Epigênese Genética/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Ribonuclease III/genética , Animais , Aquaporina 2/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/metabolismo , Regulação para Baixo , Canais Epiteliais de Sódio/metabolismo , Feminino , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA3/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/genética , Túbulos Renais Coletores/fisiologia , Masculino , Camundongos , Poliúria/genética , Poliúria/metabolismo , Proteoma , Processamento Pós-Transcricional do RNA , Reabsorção Renal , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Bioessays ; 43(1): e2000181, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33165968

RESUMO

During early embryonic development in several metazoans, accurate DNA replication is ensured by high number of replication origins. This guarantees rapid genome duplication coordinated with fast cell divisions. In Xenopus laevis embryos this program switches to one with a lower number of origins at a developmental stage known as mid-blastula transition (MBT) when cell cycle length increases and gene transcription starts. Consistent with this regulation, somatic nuclei replicate poorly when transferred to eggs, suggesting the existence of an epigenetic memory suppressing replication assembly origins at all available sites. Recently, it was shown that histone H1 imposes a non-permissive chromatin configuration preventing replication origin assembly on somatic nuclei. This somatic state can be erased by SSRP1, a subunit of the FACT complex. Here, we further develop the hypothesis that this novel form of epigenetic memory might impact on different areas of vertebrate biology going from nuclear reprogramming to cancer development.


Assuntos
Histonas , Origem de Replicação , Blástula/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética/genética , Feminino , Proteínas de Grupo de Alta Mobilidade , Histonas/genética , Histonas/metabolismo , Humanos , Gravidez , Fatores de Elongação da Transcrição
6.
Sci Rep ; 10(1): 20182, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33214630

RESUMO

Short term treatment with low doses of glucocorticoid analogues has been shown to ameliorate neurological symptoms in Ataxia-Telangiectasia (A-T), a rare autosomal recessive multisystem disease that mainly affects the cerebellum, immune system, and lungs. Molecular mechanisms underlying this clinical observation are unclear. We aimed at evaluating the effect of dexamethasone on the induction of alternative ATM transcripts (ATMdexa1). We showed that dexamethasone cannot induce an alternative ATM transcript in control and A-T lymphoblasts and primary fibroblasts, or in an ATM-knockout HeLa cell line. We also demonstrated that some of the reported readouts associated with ATMdexa1 are due to cellular artifacts and the direct induction of γH2AX by dexamethasone via DNA-PK. Finally, we suggest caution in interpreting dexamethasone effects in vitro for the results to be translated into a rational use of the drug in A-T patients.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Dexametasona/farmacologia , Processamento Alternativo/genética , Ataxia Telangiectasia/tratamento farmacológico , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Técnicas de Inativação de Genes , Células HeLa , Histonas/metabolismo , Humanos , Limite de Detecção , Fosforilação/efeitos dos fármacos
7.
Sci Rep ; 10(1): 16383, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009446

RESUMO

Nephrogenic diabetes insipidus (NDI) is a rare tubulopathy characterized by urinary concentration defect due to renal resistance to vasopressin. Loss-of-function mutations of vasopressin V2 receptor (V2R) gene (AVPR2) is the most common cause of the disease. We have identified five novel mutations L86P, R113Q, C192S, M272R, and W323_I324insR from NDI-affected patients. Functional characterization of these mutants revealed that R113Q and C192S were normally localized at the basolateral membrane of polarized Madin-Darby Canine Kidney (MDCK) cells and presented proper glycosylation maturation. On the other side, L86P, M272R, and W323_I324insR mutants were retained in endoplasmic reticulum and exhibited immature glycosylation and considerably reduced stability. All five mutants were resistant to administration of vasopressin analogues as evaluated by defective response in cAMP release. In order to rescue the function of the mutated V2R, we tested VX-809, sildenafil citrate, ibuprofen and tolvaptan in MDCK cells. Among these, tolvaptan was effective in rescuing the function of M272R mutation, by both allowing proper glycosylation maturation, membrane sorting and response to dDAVP. These results show an important proof of concept for the use of tolvaptan in patients affected by M272R mutation of V2R causing NDI.


Assuntos
Diabetes Insípido Nefrogênico/genética , Mutação/efeitos dos fármacos , Mutação/genética , Receptores de Vasopressinas/genética , Tolvaptan/farmacologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Células COS , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Chlorocebus aethiops , Cães , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Humanos , Células Madin Darby de Rim Canino , Masculino , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Vasopressinas/genética
8.
Nat Commun ; 11(1): 1345, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165637

RESUMO

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Blástula/embriologia , Blástula/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade , Histonas/química , Histonas/genética , Domínios Proteicos , Origem de Replicação , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
9.
Elife ; 92020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32163370

RESUMO

Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.


Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Diferenciação Celular , Proliferação de Células/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Quimera , Cromatografia Líquida , Clonagem Molecular , Dano ao DNA , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Espectrometria de Massas em Tandem
10.
Cell Rep ; 30(7): 2416-2429.e7, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32075739

RESUMO

It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Ligação Proteica/genética , Humanos
11.
Curr Probl Diagn Radiol ; 49(6): 398-403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31253462

RESUMO

PURPOSE: To compare magnetic resonance imaging (MRI) findings with gadoxetic acid and gadobenate dimeglumine for the diagnosis of hepatic hemangiomas. MATERIALS AND METHODS: In this retrospective study, we included 26 hemangiomas (mean size was 14 mm ± 10 mm) in 19 patients (mean age 60 ± 14 years) scanned with both gadobenate dimeglumine MRI and gadoxetic acid MRI. For each patient, we collected multiple lesion variables including location, number, size and enhancement pattern on arterial, portal venous, 3-minute and hepatobiliary phases with both gadoxetic acid and gadobenate dimeglumine. The enhancement pattern with the two contrast agents was then compared. RESULTS: The typical enhancement pattern of hepatic hemangiomas was more common-though not statistically significant-with gadobenate dimeglumine compared to gadoxetic acid (57% [15 of 26] vs 42% [11 of 26], respectively; P = 0.4057 for both peripheral globular discontinuous enhancement in the arterial phase and centripetal fill-in in the portal venous phase). A significantly higher number of hemangiomas showed centripetal fill-in or hyperintensity in the 3-minute phase with gadobenate dimeglumine compared to gadoxetic acid (88% [23 of 26) vs 58% [15 of 26]; P = 0.0266). A pseudo washout sign in the 3-minute phase was detected in one of the 5 flash-filling hemangiomas with gadoxetic acid, but not gadobenate dimeglumine. All hemangiomas were hypointense in the hepatobiliary phase with both gadobenate dimeglumine and gadoxetic acid. CONCLUSIONS: The enhancement pattern of hepatic hemangiomas may vary depending on the hepatobiliary agent, with more frequent lack of the typical pattern with gadoxetic acid compared to gadobenate dimeglumine.


Assuntos
Gadolínio DTPA/administração & dosagem , Hemangioma/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Meglumina/análogos & derivados , Compostos Organometálicos/administração & dosagem , Meios de Contraste , Feminino , Humanos , Aumento da Imagem , Interpretação de Imagem Assistida por Computador , Masculino , Meglumina/administração & dosagem , Pessoa de Meia-Idade , Estudos Retrospectivos
12.
Open Biol ; 8(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29950452

RESUMO

The development of metastatic cancer is a multistage process, which often requires decades to complete. Impairments in DNA damage control and DNA repair in cancer cell precursors generate genetically heterogeneous cell populations. However, despite heterogeneity most solid cancers have stereotypical behaviours, including invasiveness and suppression of immune responses that can be unleashed with immunotherapy targeting lymphocyte checkpoints. The mechanisms leading to the acquisition of stereotypical properties remain poorly understood. Reactivation of embryonic development processes in cells with unstable genomes might contribute to tumour expansion and metastasis formation. However, it is unclear whether these events are linked to immune response modulation. Tumours and embryos have non-self-components and need to avoid immune responses in their microenvironment. In mammalian embryos, neo-antigens are of paternal origin, while in tumour cells DNA mismatch repair and replication defects generate them. Inactivation of the maternal immune response towards the embryo, which occurs at the placental-maternal interface, is key to ensuring embryonic development. This regulation is accomplished by the trophoblast, which mimics several malignant cell features, including the ability to invade normal tissues and to avoid host immune responses, often adopting the same cancer immunoediting strategies. A better understanding as to whether and how genotoxic stress promotes cancer development through reactivation of programmes occurring during early stages of mammalian placentation could help to clarify resistance to drugs targeting immune checkpoint and DNA damage responses and to develop new therapeutic strategies to eradicate cancer.


Assuntos
Neoplasias/imunologia , Placenta/imunologia , Placentação , Animais , Microambiente Celular , Reprogramação Celular , Feminino , Humanos , Placenta/embriologia , Gravidez , Trofoblastos/imunologia
13.
Cell ; 173(6): 1508-1519.e18, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29754816

RESUMO

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.


Assuntos
Proteínas de Ciclo Celular/química , Cromátides/química , Proteínas Cromossômicas não Histona/química , DNA/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Cromatina/química , Humanos , Hidrólise , Lisina/química , Camundongos , Mutação , Proteínas Nucleares/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Nature ; 557(7703): 57-61, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29670289

RESUMO

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.


Assuntos
Replicação do DNA , Interferon Tipo I/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Citosol/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Interferon Tipo I/imunologia , Proteína Homóloga a MRE11/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , RecQ Helicases/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/deficiência
15.
Mol Cell ; 68(2): 414-430.e8, 2017 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-29053959

RESUMO

To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.


Assuntos
Proteína BRCA2/deficiência , Quebras de DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Humanos , Proteína Homóloga a MRE11 , Fatores de Transcrição/genética
16.
Cell Rep ; 21(2): 333-340, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-29020621

RESUMO

Fanconi anemia (FA) is a genetic disorder characterized by a defect in DNA interstrand crosslink (ICL) repair, chromosomal instability, and a predisposition to cancer. Recently, two RAD51 mutations were reported to cause an FA-like phenotype. Despite the tight association of FA/HR proteins with replication fork (RF) stabilization during normal replication, it remains unknown how FA-associated RAD51 mutations affect replication beyond ICL lesions. Here, we report that these mutations fail to protect nascent DNA from MRE11-mediated degradation during RF stalling in Xenopus laevis egg extracts. Reconstitution of DNA protection in vitro revealed that the defect arises directly due to altered RAD51 properties. Both mutations induce pronounced structural changes and RAD51 filament destabilization that is not rescued by prevention of ATP hydrolysis due to aberrant ATP binding. Our results further interconnect the FA pathway with DNA replication and provide mechanistic insight into the role of RAD51 in recombination-independent mechanisms of genome maintenance.


Assuntos
Replicação do DNA , Anemia de Fanconi/genética , Mutação , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Proteína Homóloga a MRE11/metabolismo , Ligação Proteica , Estabilidade Proteica , Rad51 Recombinase/genética , Xenopus
17.
Prog Mol Subcell Biol ; 56: 515-539, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28840251

RESUMO

The centromere is the genetic locus that specifies the site of kinetochore assembly, where the chromosome will attach to the kinetochore microtubule. The pericentromere is the physical region responsible for the geometry of bi-oriented sister kinetochores in metaphase. In budding yeast the 125 bp point centromere is sufficient to specify kinetochore assembly. The flanking region is enriched (3X) in cohesin and condensin relative to the remaining chromosome arms. The enrichment spans about 30-50 kb around each centromere. We refer to the flanking chromatin as the pericentromere in yeast. In mammals, a 5-10 Mb region dictates where the kinetochore is built. The kinetochore interacts with a very small fraction of DNA on the surface of the centromeric region. The remainder of the centromere lies between the sister kinetochores. This is typically called centromere chromatin. The chromatin sites that directly interface to microtubules cannot be identified due to the repeated sequence within the mammalian centromere. However in both yeast and mammals, the total amount of DNA between the sites of microtubule attachment in metaphase is highly conserved. In yeast the 16 chromosomes are clustered into a 250 nm diameter region, and 800 kb (16 × 50 kb) or ~1 Mb of DNA lies between sister kinetochores. In mammals, 5-10 Mb lies between sister kinetochores. In both organisms the sister kinetochores are separated by about 1 µm. Thus, centromeres of different organisms differ in how they specify kinetochore assembly, but there may be important centromere chromatin functions that are conserved throughout phylogeny. Recently, centromeric chromatin has been reconstituted in vitro using alpha satellite DNA revealing unexpected features of centromeric DNA organization, replication, and response to stress. We will focus on the conserved features of centromere in this review.


Assuntos
Centrômero/química , Centrômero/metabolismo , Animais , Cromatina , Cinetocoros , Microtúbulos , Saccharomyces cerevisiae
18.
Mol Cell ; 67(5): 867-881.e7, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28757209

RESUMO

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.


Assuntos
Proteína BRCA2/metabolismo , Replicação do DNA , DNA/biossíntese , Rad51 Recombinase/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteína BRCA2/genética , Sítios de Ligação , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Instabilidade Genômica , Humanos , Proteína Homóloga a MRE11 , Masculino , Mutação , Ligação Proteica , Rad51 Recombinase/genética , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Proteínas de Xenopus/genética , Xenopus laevis/genética
19.
Methods Enzymol ; 591: 211-232, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28645370

RESUMO

Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. The possibility of manipulating the protein content by using protein depletion procedures makes egg extract a powerful system to study proteins whose inactivation results in cellular lethality. The egg extract has been increasingly used to study DNA damage response and the coordination of DNA replication with DNA repair. The recent development of advanced imaging techniques based on electron microscopy has allowed the characterization of replication intermediates formed in the absence of essential DNA repair proteins. These studies have been important to understand how cells maintain genome stability under unchallenged and stressful conditions. Here, we present a collection of protocols that have been developed to recapitulate DNA damage response activated by chromosome breakage in egg extract and to isolate replication intermediates for electron microscopy analysis using sperm nuclei or more defined genomic substrates.


Assuntos
Dano ao DNA , Replicação do DNA , Modelos Biológicos , Animais , Cromatina/metabolismo , Óvulo/citologia , Xenopus laevis
20.
EMBO Rep ; 18(6): 1000-1012, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28389464

RESUMO

Camptothecin-induced locking of topoisomerase 1 on DNA generates a physical barrier to replication fork progression and creates topological stress. By allowing replisome rotation, absence of the Tof1/Csm3 complex promotes the conversion of impending topological stress to DNA catenation and causes camptothecin hypersensitivity. Through synthetic viability screening, we discovered that histone H4 K16 deacetylation drives the sensitivity of yeast cells to camptothecin and that inactivation of this pathway by mutating H4 K16 or the genes SIR1-4 suppresses much of the hypersensitivity of tof1∆ strains towards this agent. We show that disruption of rDNA or telomeric silencing does not mediate camptothecin resistance but that disruption of Sir1-dependent chromatin domains is sufficient to suppress camptothecin sensitivity in wild-type and tof1∆ cells. We suggest that topoisomerase 1 inhibition in proximity of these domains causes topological stress that leads to DNA hypercatenation, especially in the absence of the Tof1/Csm3 complex. Finally, we provide evidence of the evolutionarily conservation of this mechanism.


Assuntos
Camptotecina/farmacologia , Cromatina , Proteínas de Saccharomyces cerevisiae/metabolismo , Benzamidas/farmacologia , Camptotecina/metabolismo , Proteínas de Ciclo Celular , Dano ao DNA , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Fúngico/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Naftóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo
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