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1.
J Biol Chem ; 300(6): 107368, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38750793

RESUMO

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.


Assuntos
Diester Fosfórico Hidrolases , Humanos , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Cristalografia por Raios X , Biologia Computacional/métodos , Motivos de Ligação ao RNA/genética
2.
Nucleic Acids Res ; 52(5): 2416-2433, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38224455

RESUMO

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.


Assuntos
Enzimas Reparadoras do DNA , Fosfotransferases (Aceptor do Grupo Álcool) , Animais , Humanos , Camundongos , Acetilação , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Mamíferos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Polinucleotídeo 5'-Hidroxiquinase/genética
3.
Nat Commun ; 14(1): 8169, 2023 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-38071370

RESUMO

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5'-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease.


Assuntos
COVID-19 , DNA Glicosilases , Cricetinae , Animais , Humanos , COVID-19/genética , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Enzima de Conversão de Angiotensina 2/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Genoma , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo
4.
Int J Mol Sci ; 24(18)2023 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-37762489

RESUMO

Base excision repair (BER) corrects forms of oxidative, deamination, alkylation, and abasic single-base damage that appear to have minimal effects on the helix. Since its discovery in 1974, the field has grown in several facets: mechanisms, biology and physiology, understanding deficiencies and human disease, and using BER genes as potential inhibitory targets to develop therapeutics. Within its segregation of short nucleotide (SN-) and long patch (LP-), there are currently six known global mechanisms, with emerging work in transcription- and replication-associated BER. Knockouts (KOs) of BER genes in mouse models showed that single glycosylase knockout had minimal phenotypic impact, but the effects were clearly seen in double knockouts. However, KOs of downstream enzymes showed critical impact on the health and survival of mice. BER gene deficiency contributes to cancer, inflammation, aging, and neurodegenerative disorders. Medicinal targets are being developed for single or combinatorial therapies, but only PARP and APE1 have yet to reach the clinical stage.


Assuntos
Medicina , Humanos , Animais , Camundongos , Camundongos Knockout , Envelhecimento , Reparo do DNA , Biologia
5.
bioRxiv ; 2023 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-37645927

RESUMO

Mammalian polynucleotide kinase 3'-phosphatase (PNKP) is a dual-function DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, which generate 3'-OH and 5'-phosphate termini respectively, as substrates for DNA polymerase and DNA ligase to complete DNA repair. PNKP is thus involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes, which involve distinct sets of proteins. In this study, we report that PNKP is acetylated at two lysine (K142 and K226) residues. While K142 (AcK142) is constitutively acetylated by p300, CBP acetylates K226 (AcK226) only after DSB induction. Co-immunoprecipitation analysis using antibodies specific for PNKP peptides containing AcK142 or AcK226 of PNKP showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP only with DSBR proteins. Although acetylation at these residues did not significantly affect the enzymatic activity of PNKP in vitro, cells expressing nonacetylable PNKP (K142R or K226R) accumulated DNA damage, specifically in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This observation is consistent with the reported degradation of CBP but not p300 in HD cells. Moreover, genomes of HD cells progressively accumulated DSBs specifically in the transcribed genes. Chromatin-immunoprecipitation analysis using anti-AcK142 or anti-AcK226 antibodies demonstrated an association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings collectively demonstrate that acetylation at two lysine residues located in different domains of PNKP regulates its functionally distinct role in BER/SSBR vs. DSBR.

6.
Methods Mol Biol ; 2701: 149-156, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37574480

RESUMO

R loops (DNA-RNA hybrid) are three-stranded nucleic acid structures that comprise of template DNA strand hybridized with the nascent RNA leaving the displaced non-template strand. Although a programmed R loop formation can serve as powerful regulators of gene expression, these structures can also turn into major sources of genomic instability and contribute to the development of diseases. Therefore, understanding how cells prevent the deleterious consequences of R loops yet allow R loop formation to participate in various physiological processes will help to understand how their homeostasis is maintained. Detection and quantitative measurements of R loops are critical that largely relied on S9.6 antibody. Immunofluorescence methods are frequently used to localize and quantify R loops in the cell but they require specialized tools for analysis and relatively expensive; therefore, they are not always useful for initial assessments of R loop accumulation. Here, we describe an improved slot blot protocol to detect and estimate R loops and show its sensitivity and specificity using the S9.6 antibody. Since specific factors protecting cells from harmful R loop accumulation are expanding, this protocol can be used to determine R loop accumulation in research and clinical settings.


Assuntos
Estruturas R-Loop , RNA , Humanos , Conformação de Ácido Nucleico , RNA/genética , DNA/genética , Anticorpos/química , Instabilidade Genômica
7.
J Biol Chem ; 299(5): 104714, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37061005

RESUMO

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.


Assuntos
Enzimas Reparadoras do DNA , Leucócitos Mononucleares , Fosfotransferases (Aceptor do Grupo Álcool) , Ataxias Espinocerebelares , Humanos , DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Leucócitos Mononucleares/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ataxias Espinocerebelares/genética
8.
J Biol Chem ; 299(3): 102991, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36758800

RESUMO

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.


Assuntos
Quebras de DNA de Cadeia Dupla , Escherichia coli , Animais , Humanos , Escherichia coli/genética , Reparo do DNA , Reparo do DNA por Junção de Extremidades , DNA , RNA/genética , DNA Ligase Dependente de ATP , Mamíferos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Enzimas Reparadoras do DNA/genética
9.
PLoS One ; 17(5): e0267839, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35576221

RESUMO

Thirdhand smoke (THS) is a newly described health hazard composed of toxicants, mutagens and carcinogens, including nicotine-derived tobacco specific nitrosamines (TSNAs), one of which is 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA). Although TSNAs are generally potent carcinogens, the risk of NNA, which is specific to THS, is poorly understood. We recently reported that THS exposure-induced adverse impact on DNA replication and transcription with implications in the development of cancer and other diseases. Here, we investigated the role of NNA in THS exposure-induced harmful effects on fundamental cellular processes. We exposed cultured human lung epithelial BEAS-2B cells to NNA. The formation of DNA base damages was assessed by Long Amplicon QPCR (LA-QPCR); DNA double-strand breaks (DSBs) and NNA effects on replication and transcription by immunofluorescence (IF); and genomic instability by micronuclei (MN) formation. We found increased accumulation of oxidative DNA damage and DSBs as well as activation of DNA damage response pathway, after exposure of cells to NNA. Impaired S phase progression was also evident. Consistent with these results, we found increased MN formation, a marker of genomic instability, in NNA-exposed cells. Furthermore, ongoing RNA synthesis was significantly reduced by NNA exposure, however, RNA synthesis resumed fully after a 24h recovery period only in wild-type cells but not in those deficient in transcription-coupled nucleotide excision repair (TC-NER). Importantly, these cellular effects are common with the THS-exposure induced effects. Our findings suggest that NNA in THS could be a contributing factor for THS exposure-induced adverse health effect.


Assuntos
Nitrosaminas , Poluição por Fumaça de Tabaco , Aldeídos , Carcinógenos/análise , DNA , Dano ao DNA , Instabilidade Genômica , Humanos , Pulmão/metabolismo , Nicotina/análise , Nitrosaminas/análise , Nitrosaminas/toxicidade , Piridinas , RNA , Nicotiana/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Poluição por Fumaça de Tabaco/análise
10.
Biophys J ; 120(15): 3152-3165, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34197805

RESUMO

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Modelos Moleculares , RNA Viral/genética , Espalhamento a Baixo Ângulo , Proteínas não Estruturais Virais , Replicação Viral , Difração de Raios X
11.
Prog Biophys Mol Biol ; 164: 72-80, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33753087

RESUMO

Cell survival largely depends on the faithful maintenance of genetic material since genomic DNA is constantly exposed to genotoxicants from both endogenous and exogenous sources. The evolutionarily conserved base excision repair (BER) pathway is critical for maintaining genome integrity by eliminating highly abundant and potentially mutagenic oxidized DNA base lesions. BER is a multistep process, which is initiated with recognition and excision of the DNA base lesion by a DNA glycosylase, followed by DNA end processing, gap filling and finally sealing of the nick. Besides genome maintenance by global BER, DNA glycosylases have been found to play additional roles, including preferential repair of oxidized lesions from transcribed genes, modulation of the immune response, participation in active DNA demethylation and maintenance of the mitochondrial genome. Central to these functions is the DNA glycosylase NEIL2. Its loss results in increased accumulation of oxidized base lesions in the transcribed genome, triggers an immune response and causes early neurodevelopmental defects, thus emphasizing the multitasking capabilities of this repair protein. Here we review the specialized functions of NEIL2 and discuss the consequences of its absence both in vitro and in vivo.


Assuntos
DNA Glicosilases , Animais , DNA , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos
12.
Structure ; 29(1): 1-2, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33417890

RESUMO

The canonical DNA glycosylase role is global base damage repair but includes functions in epigenetic gene regulation, immune response modulation, replication, and transcription. In this issue of Structure, Eckenroth et al. (2020) present the NEIL2 glycosylase structure. Its catalytic domain flexibility differentiates it from most other glycosylases and suggests novel regulatory mechanisms.


Assuntos
DNA Glicosilases , Animais , DNA , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(25): 14127-14138, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522879

RESUMO

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.


Assuntos
Síndrome de Cockayne/genética , Proteínas de Ligação a DNA/química , Endonucleases/química , Proteínas Nucleares/química , Mutação Puntual , Fatores de Transcrição/química , Xeroderma Pigmentoso/genética , Sítios de Ligação , Sequência Conservada , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Environ Mol Mutagen ; 61(6): 635-646, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32267018

RESUMO

Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to the development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here, we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1, and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.


Assuntos
Poluição do Ar em Ambientes Fechados/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Transcrição Gênica/efeitos dos fármacos , Poluentes Atmosféricos/toxicidade , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Humanos , Testes para Micronúcleos , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
15.
Proc Natl Acad Sci U S A ; 117(14): 8154-8165, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32205441

RESUMO

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.


Assuntos
Ataxina-3/genética , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Doença de Machado-Joseph/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Ataxina-3/metabolismo , Encéfalo/patologia , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Drosophila , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Peptídeos/genética , RNA Interferente Pequeno/metabolismo
17.
Nucleic Acids Res ; 46(9): 4515-4532, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29522130

RESUMO

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.


Assuntos
Transformação Celular Neoplásica , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Instabilidade Genômica , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Dano ao DNA , Replicação do DNA , Desoxirribonuclease (Dímero de Pirimidina)/genética , Células Epiteliais/enzimologia , Humanos , Neoplasias Pulmonares/enzimologia , Mutação , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologia
18.
Clin Sci (Lond) ; 132(4): 475-488, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29440622

RESUMO

Exposure to thirdhand smoke (THS) is a recently described health concern that arises in many indoor environments. However, the carcinogenic potential of THS, a critical consideration in risk assessment, remains untested. Here we investigated the effects of short-term early exposure to THS on lung carcinogenesis in A/J mice. Forty weeks after THS exposure from 4 to 7 weeks of age, the mice had increased incidence of lung adenocarcinoma, tumor size and, multiplicity, compared with controls. In vitro studies using cultured human lung cancer cells showed that THS exposure induced DNA double-strand breaks and increased cell proliferation and colony formation. RNA sequencing analysis revealed that THS exposure induced endoplasmic reticulum stress and activated p53 signaling. Activation of the p53 pathway was confirmed by an increase in its targets p21 and BAX. These data indicate that early exposure to THS is associated with increased lung cancer risk.


Assuntos
Neoplasias Pulmonares/induzido quimicamente , Fumar/efeitos adversos , Fatores de Tempo , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Incidência , Camundongos , Nicotiana/efeitos adversos
19.
Nat Commun ; 8: 15855, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28653660

RESUMO

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via 'phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.


Assuntos
DNA/genética , Endonucleases Flap/metabolismo , Instabilidade Genômica , Fosfatos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , DNA/metabolismo , Reparo do DNA , Replicação do DNA , Endonucleases Flap/química , Endonucleases Flap/genética , Humanos , Mutação , Fosfatos/química , Alinhamento de Sequência , Especificidade por Substrato
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