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1.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372559

RESUMO

The human BK polyomavirus (BKPyV) is latent in the kidneys of most adults, but can be reactivated in immunosuppressed states, such as following renal transplantation. If left unchecked, BK polyomavirus nephropathy (PyVAN) and possible graft loss may result from viral destruction of tubular epithelial cells and interstitial fibrosis. When coupled with regular post-transplant screening, immunosuppression reduction has been effective in limiting BKPyV viremia and the development of PyVAN. Antiviral drugs that are safe and effective in combating BKPyV have not been identified but would be a benefit in complementing or replacing immunosuppression reduction. The present study explores inhibition of the host DNA damage response (DDR) as an antiviral strategy. Immunohistochemical and immunofluorescent analyses of PyVAN biopsies provide evidence for stimulation of a DDR in vivo. DDR pathways were also stimulated in vitro following BKPyV infection of low-passage human renal proximal tubule epithelial cells. The role of Chk1, a protein kinase known to be involved in the replication stress-induced DDR, was examined by inhibition with the small molecule LY2603618 and by siRNA-mediated knockdown. Inhibition of Chk1 resulted in decreased replication of BKPyV DNA and viral spread. Activation of mitotic pathways was associated with the reduction in BKPyV replication. Chk1 inhibitors that are found to be safe and effective in clinical trials for cancer should also be evaluated for antiviral activity against BKPyV.


Assuntos
Vírus BK/genética , Quinase 1 do Ponto de Checagem/metabolismo , Infecções por Polyomavirus/tratamento farmacológico , Vírus BK/patogenicidade , Células Cultivadas , Quinase 1 do Ponto de Checagem/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Rim/patologia , Rim/virologia , Transplante de Rim , Compostos de Fenilureia/farmacologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/imunologia , Pirazinas/farmacologia , Infecções Tumorais por Vírus/tratamento farmacológico , Infecções Tumorais por Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
2.
Development ; 148(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34345913

RESUMO

STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.


Assuntos
Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Reparo do DNA/fisiologia , Feminino , Imunoprecipitação/métodos , Masculino , Camundongos , Neurogênese/fisiologia , Neurônios/metabolismo , Gravidez
3.
Biomolecules ; 11(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34356684

RESUMO

The maintenance of genome integrity in the cell is an essential process for the accurate transmission of the genetic material. BRCA2 participates in this process at several levels, including DNA repair by homologous recombination, protection of stalled replication forks, and cell division. These activities are regulated and coordinated via cell-cycle dependent modifications. Pathogenic variants in BRCA2 cause genome instability and are associated with breast and/or ovarian cancers. BRCA2 is a very large protein of 3418 amino acids. Most well-characterized variants causing a strong predisposition to cancer are mutated in the C-terminal 700 residues DNA binding domain of BRCA2. The rest of the BRCA2 protein is predicted to be disordered. Interactions involving intrinsically disordered regions (IDRs) remain difficult to identify both using bioinformatics tools and performing experimental assays. However, the lack of well-structured binding sites provides unique functional opportunities for BRCA2 to bind to a large set of partners in a tightly regulated manner. We here summarize the predictive and experimental arguments that support the presence of disorder in BRCA2. We describe how BRCA2 IDRs mediate self-assembly and binding to partners during DNA double-strand break repair, mitosis, and meiosis. We highlight how phosphorylation by DNA repair and cell-cycle kinases regulate these interactions. We finally discuss the impact of cancer-associated variants on the function of BRCA2 IDRs and more generally on genome stability and cancer risk.


Assuntos
Proteína BRCA2/química , Proteína BRCA2/metabolismo , Reparo do DNA/fisiologia , Proteína BRCA2/genética , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Feminino , Humanos , Interfase/fisiologia , Espectroscopia de Ressonância Magnética , Mitose , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo
4.
Sci Rep ; 11(1): 16354, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381129

RESUMO

Stroke is a common cause of death worldwide and leads to disability and cognitive dysfunction. Ischemic stroke and hemorrhagic stroke are major categories of stroke, accounting for 68% and 32% of strokes, respectively. Each year, 15 million people experience stroke worldwide, and the stroke incidence is rising. Epigenetic modifications regulate gene transcription and play a major role in stroke. Accordingly, histone deacetylase 1 (HDAC1) participates in DNA damage repair and cell survival. However, the mechanisms underlying the role of HDAC1 in stroke pathogenesis are still controversial. Therefore, we investigated the role of HDAC1 in stroke by using a rat model of endothelin-1-induced brain ischemia. Our results revealed that HDAC1 was deregulated following stroke, and its expressional level and enzymatic activity were decreased. We also used MS-275 to inhibit HDAC1 function in rats exposed to ischemic insult. We found that HDAC1 inhibition promoted the infarct volume, neuronal loss, DNA damage, neuronal apoptosis after stroke, and levels of reactive oxygen species and inflammation cytokines. Additionally, HDAC1 inhibition deteriorated the behavioral outcomes of rats with ischemic insult. Overall, our findings demonstrate that HDAC1 participates in ischemic pathogenesis in the brain and possesses potential for use as a therapeutic target.


Assuntos
Histona Desacetilase 1/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Animais , Apoptose/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Sobrevivência Celular/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Epigênese Genética/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Ratos , Ratos Sprague-Dawley
5.
Nat Commun ; 12(1): 4108, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226550

RESUMO

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.


Assuntos
DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Triagem , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Glicosilases/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Especificidade por Substrato
6.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205449

RESUMO

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5',8-cyclo-2'-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction-modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme's recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5'S)-5',8-cyclo-2'-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5'R)-5',8-cyclo-2'-deoxyadenosine (RcdA). Interestingly, in the case of 5',8-cyclo-2'-deoxyguanosine (cdG), both 5'S and 5'R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.


Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Animais , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Oligonucleotídeos/metabolismo
7.
Int J Mol Sci ; 22(13)2021 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-34281248

RESUMO

Age-related macular degeneration (AMD), the main cause of vision loss in the elderly, is associated with oxidation in the retina cells promoting telomere attrition. Activation of telomerase was reported to improve macular functions in AMD patients. The catalytic subunit of human telomerase (hTERT) may directly interact with proteins important for senescence, DNA damage response, and autophagy, which are impaired in AMD. hTERT interaction with mTORC1 (mTOR (mechanistic target of rapamycin) complex 1) and PINK1 (PTEN-induced kinase 1) activates macroautophagy and mitophagy, respectively, and removes cellular debris accumulated over AMD progression. Ectopic expression of telomerase in retinal pigment epithelium (RPE) cells lengthened telomeres, reduced senescence, and extended their lifespan. These effects provide evidence for the potential of telomerase in AMD therapy. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may be involved in AMD pathogenesis through decreasing oxidative stress and senescence, regulation of vascular endothelial growth factor (VEGF), and improving autophagy. PGC-1α and TERT form an inhibitory positive feedback loop. In conclusion, telomerase activation and its ectopic expression in RPE cells, as well as controlled clinical trials on the effects of telomerase activation in AMD patients, are justified and should be assisted by PGC-1α modulators to increase the therapeutic potential of telomerase in AMD.


Assuntos
Degeneração Macular/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Telomerase/metabolismo , Envelhecimento/metabolismo , Autofagia/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Degeneração Macular/fisiopatologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais , Telomerase/fisiologia , Telômero/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203408

RESUMO

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias do Endométrio/metabolismo , Mutação/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Neoplasias do Endométrio/genética , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Células MCF-7 , Reação em Cadeia da Polimerase , Polinucleotídeo Adenililtransferase/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
9.
Mol Cell Biol ; 41(10): e0008121, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34251884

RESUMO

Cullin-4 ubiquitin ligase (CRL4) complexes are differentially composed and highly dynamic protein assemblies that control many biological processes, including the global genome nucleotide excision repair (GG-NER) pathway. Here, we identified the kinase mitogen-activated protein kinase kinase kinase 1 (MEKK1) as a novel constitutive interactor of a cytosolic CRL4 complex that disassembles after DNA damage due to the caspase-mediated cleavage of MEKK1. The kinase activity of MEKK1 was important to trigger autoubiquitination of the CRL4 complex by K48- and K63-linked ubiquitin chains. MEKK1 knockdown prohibited DNA damage-induced degradation of the CRL4 component DNA-damage binding protein 2 (DDB2) and the CRL4 substrate p21 and also cell recovery and survival. A ubiquitin replacement strategy revealed a contribution of K63-branched ubiquitin chains for DNA damage-induced DDB2/p21 decay, cell cycle regulation, and cell survival. These data might also have implications for cancer, as frequently occurring mutations of MEKK1 might have an impact on genome stability and the therapeutic efficacy of CRL4-dependent immunomodulatory drugs such as thalidomide derivatives.


Assuntos
Reparo do DNA/fisiologia , MAP Quinase Quinase Quinase 1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA/química , Dano ao DNA/fisiologia , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , MAP Quinase Quinase Quinase 1/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Ubiquitinação
10.
PLoS Genet ; 17(7): e1009715, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329293

RESUMO

Histone methylation is dynamically regulated to shape the epigenome and adjust central nuclear processes including transcription, cell cycle control and DNA repair. Lysine-specific histone demethylase 2 (LSD2) has been implicated in multiple types of human cancers. However, its functions remain poorly understood. This study investigated the histone demethylase LSD2 homolog AMX-1 in C. elegans and uncovered a potential link between H3K4me2 modulation and DNA interstrand crosslink (ICL) repair. AMX-1 is a histone demethylase and mainly localizes to embryonic cells, the mitotic gut and sheath cells. Lack of AMX-1 expression resulted in embryonic lethality, a decreased brood size and disorganized premeiotic tip germline nuclei. Expression of AMX-1 and of the histone H3K4 demethylase SPR-5 is reciprocally up-regulated upon lack of each other and the mutants show increased H3K4me2 levels in the germline, indicating that AMX-1 and SPR-5 regulate H3K4me2 demethylation. Loss of AMX-1 function activates the CHK-1 kinase acting downstream of ATR and leads to the accumulation of RAD-51 foci and increased DNA damage-dependent apoptosis in the germline. AMX-1 is required for the proper expression of mismatch repair component MutL/MLH-1 and sensitivity against ICLs. Interestingly, formation of ICLs lead to ubiquitination-dependent subcellular relocalization of AMX-1. Taken together, our data suggest that AMX-1 functions in ICL repair in the germline.


Assuntos
Reparo do DNA/genética , Histona Desmetilases/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Núcleo Celular/metabolismo , Dano ao DNA/genética , Reparo do DNA/fisiologia , Células Germinativas/metabolismo , Histona Desmetilases/fisiologia , Histonas/genética , Metilação , Processamento de Proteína Pós-Traducional/genética , Ubiquitinação
11.
PLoS Genet ; 17(7): e1009459, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228704

RESUMO

Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination.


Assuntos
Reparo do DNA/fisiologia , Histona Acetiltransferases/genética , Recombinação Homóloga , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transativadores/genética , Acetilação , Cromatina/genética , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Histona Acetiltransferases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo
12.
Cells ; 10(6)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203749

RESUMO

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.


Assuntos
Reparo do DNA/genética , Reparo do DNA/fisiologia , RNA Longo não Codificante/fisiologia , Animais , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/fisiologia , Reparo do DNA por Junção de Extremidades/fisiologia , Instabilidade Genômica , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Reparo de DNA por Recombinação/fisiologia
13.
Dev Cell ; 56(15): 2207-2222.e7, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34256011

RESUMO

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.


Assuntos
Apoptose/fisiologia , Reparo do DNA/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Animais , Proteína Adaptadora de Sinalização CRADD/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Células HeLa , Humanos , Ubiquitinação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
14.
Nat Protoc ; 16(8): 3933-3953, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34163064

RESUMO

The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing. We also describe complementation with orthogonal DSB repair assays and omics analyses to validate the reporter readouts. The bioluminescent repair reporter system provides longitudinal and rapid readout (~seconds per sample) to accurately and efficiently measure the efficacy of genome-editing tools and small-molecule modulators on DSB repair. This protocol takes ~2-4 weeks to establish, and as little as 2 h to complete the assay. The entire bioluminescent repair reporter procedure can be performed by one person with standard molecular biology expertise and equipment. However, orthogonal DNA repair assays would require a specialized facility that performs Sanger sequencing or next-generation sequencing.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Medições Luminescentes/métodos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais
15.
Mutat Res Rev Mutat Res ; 787: 108347, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34083046

RESUMO

APE2 is a rising vital player in the maintenance of genome and epigenome integrity. In the past several years, a series of studies have shown the critical roles and functions of APE2. We seek to provide the first comprehensive review on several aspects of APE2 in genome and epigenome integrity. We first summarize the distinct functional domains or motifs within APE2 including EEP (endonuclease/exonuclease/phosphatase) domain, PIP box and Zf-GRF motifs from eight species (i.e., Homo sapiens, Mus musculus, Xenopus laevis, Ciona intestinalis, Arabidopsis thaliana, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Trypanosoma cruzi). Then we analyze various APE2 nuclease activities and associated DNA substrates, including AP endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-5' exonuclease activities. We also examine several APE2 interaction proteins, including PCNA, Chk1, APE1, Myh1, and homologous recombination (HR) factors such as Rad51, Rad52, BRCA1, BRCA2, and BARD1. Furthermore, we provide insights into the roles of APE2 in various DNA repair pathways (base excision repair, single-strand break repair, and double-strand break repair), DNA damage response (DDR) pathways (ATR-Chk1 and p53-dependent), immunoglobulin class switch recombination and somatic hypermutation, as well as active DNA demethylation. Lastly, we summarize critical functions of APE2 in growth, development, and diseases. In this review, we provide the first comprehensive perspective which dissects all aspects of the multiple-function protein APE2 in genome and epigenome integrity.


Assuntos
Proteínas de Arabidopsis/genética , Endonucleases/genética , Animais , Proteínas de Arabidopsis/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Desmetilação do DNA , Reparo do DNA/genética , Reparo do DNA/fisiologia , Epigenoma/genética , Humanos , Imunidade/genética , Imunidade/fisiologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
Mutat Res Rev Mutat Res ; 787: 108359, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34083047

RESUMO

Genome instability denotes an increased tendency to alterations in the genome during cell life cycle, driven by a large variety of endogenous and exogenous insults. Ageing is characterized by the presence of damage to various cellular constituents, but genome alterations, randomly accumulating with age in different tissues, constitute the key target in this process, and are believed to be the main factor of ageing. Age-related failure of DNA repair pathways allows DNA lesions to occur more frequently, and their accumulation over time contributes to the age-associated decrease in genome integrity in somatic cells. The micronucleus (MN) test is one of the most widely used assays to evaluate genomic instability in different surrogate tissues. A large number of studies has consistently shown a progressive increase in MN frequency with age, starting from very young age groups onwards. Therefore, MN frequency is a suitable biomarker of genomic instability in ageing. Frailty is a multidimensional geriatric syndrome of unsuccessful ageing, characterized by decreased biological reserves and increased vulnerability to external stressors, involving a higher risk of negative health outcomes. Although there is a well-founded belief that genome instability is involved in the frailty syndrome, only two studies investigated the relationship between MN frequency and frailty, not allowing to draw a definite conclusion on the utility of this biomarker for frailty detection. The use of MN and other genomic biomarkers in the detection and follow-up of patients affected by or at risk of frailty has the potential to accumulate evidence on the clinical impact of this approach in the identification and control of frailty in older people.


Assuntos
Instabilidade Genômica/genética , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Feminino , Idoso Fragilizado , Humanos , Masculino , Testes para Micronúcleos/métodos
17.
Mutat Res Rev Mutat Res ; 787: 108362, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34083050

RESUMO

Radiotherapy is one of the primary modalities for cancer treatment, and its efficiency usually relies on cellular radiosensitivity. DNA damage repair is a core content of cellular radiosensitivity, and the primary mechanism of which includes non-homologous end-joining (NHEJ) and homologous recombination (HR). By affecting DNA damage repair, histone methylation regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs) participates in the regulation of cellular radiosensitivity via three mechanisms: (a) recruiting DNA repair-related proteins, (b) regulating the expressions of DNA repair genes, and (c) mediating the dynamic change of chromatin. Interestingly, both aberrantly high and low levels of histone methylation could impede DNA repair processes. Here we reviewed the mechanisms of the dual effects of histone methylation on cell response to radiation. Since some inhibitors of HMTs and HDMs are reported to increase cellular radiosensitivity, understanding their molecular mechanisms may be helpful in developing new drugs for the therapy of radioresistant tumors.


Assuntos
Histona Desmetilases/metabolismo , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Feminino , Histona Desmetilases/genética , Histona Metiltransferases/genética , Histonas/genética , Humanos , Masculino , Tolerância a Radiação
18.
Exp Cell Res ; 405(2): 112679, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34102225

RESUMO

DNA damage is a constant stressor to the cell. Persistent damage to the DNA over time results in an increased risk of mutation and an accumulation of mutations with age. Loss of efficient DNA damage repair can lead to accelerated ageing phenotypes or an increased cancer risk, and the trade-off between cancer susceptibility and longevity is often driven by the cell's response to DNA damage. High levels of mutations in DNA repair mutants often leads to excessive cell death and stem cell exhaustion which may promote premature ageing. Stem cells themselves have distinct characteristics that enable them to retain low mutation rates. However, when mutations do arise, stem cell clonal expansion can also contribute to age-related tissue dysfunction as well as heightened cancer risk. In this review, we will highlight increasing DNA damage and mutation accumulation as hallmarks common to both ageing and cancer. We will propose that anti-ageing interventions might be cancer preventative and discuss the mechanisms through which they may act.


Assuntos
Envelhecimento/genética , Dano ao DNA/genética , Reparo do DNA/genética , Instabilidade Genômica/genética , Longevidade/genética , Reparo do DNA/fisiologia , Neoplasias/genética
19.
Sci Rep ; 11(1): 13077, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158547

RESUMO

Ulcerative colitis (UC) is a DNA damage-associated chronic inflammatory disease; the DNA double-strand break (DSB) repair pathway participates in UC-associated dysplasia/colitic cancer carcinogenesis. The DSB/interferon regulatory factor-1 (IRF-1) pathway can induce PD-L1 expression transcriptionally. However, the association of PD-L1/DSB/IRF-1 with sporadic colorectal cancer (SCRC), and UC-associated dysplasia/colitic cancer, remains elusive. Therefore, we investigated the significance of the PD-L1/DSB repair pathway using samples from 17 SCRC and 12 UC patients with rare UC-associated dysplasia/colitic cancer cases by immunohistochemical analysis. We compared PD-L1 expression between patients with SCRC and UC-associated dysplasia/colitic cancer and determined the association between PD-L1 and the CD8+ T-cell/DSB/IRF-1 axis in UC-associated dysplasia/colitic cancer. PD-L1 expression in UC and UC-associated dysplasia/colitic cancer was higher than in normal mucosa or SCRC, and in CD8-positive T lymphocytes in UC-associated dysplasia/colitic cancer than in SCRC. Moreover, PD-L1 upregulation was associated with γH2AX (DSB marker) and IRF-1 upregulation in UC-associated dysplasia/colitic cancer. IRF-1 upregulation was associated with γH2AX upregulation in UC-associated dysplasia/colitic cancer but not in SCRC. Multicolour immunofluorescence staining validated γH2AX/IRF-1/PD-L1 co-expression in colitic cancer tissue sections. Thus, immune cell-induced inflammation might activate the DSB/IRF-1 axis, potentially serving as the primary regulatory mechanism of PD-L1 expression in UC-associated carcinogenesis.


Assuntos
Antígeno B7-H1/genética , Neoplasias do Colo/genética , Reparo do DNA/genética , Adulto , Idoso , Antígeno B7-H1/metabolismo , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Feminino , Expressão Gênica , Humanos , Fator Regulador 1 de Interferon/genética , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Ativação Transcricional
20.
Biochemistry ; 60(23): 1797-1807, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34080848

RESUMO

DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.


Assuntos
Citosina/análogos & derivados , Reparo do DNA/fisiologia , DNA/química , Arginina/química , Sequência de Bases/genética , Citosina/química , Adutos de DNA/química , Dano ao DNA/fisiologia , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Histonas/metabolismo , Humanos , Cinética , Lisina/química , Mutação/genética , Peptídeos/química
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