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1.
Cell ; 145(2): 198-211, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21496641

RESUMEN

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.


Asunto(s)
Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , ADN/metabolismo , Análisis Mutacional de ADN , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Especificidad por Sustrato
2.
Nucleic Acids Res ; 52(5): 2416-2433, 2024 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-38224455

RESUMEN

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.


Asunto(s)
Enzimas Reparadoras del ADN , Fosfotransferasas (Aceptor de Grupo Alcohol) , Animales , Humanos , Ratones , Acetilación , Daño del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Mamíferos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Polinucleótido 5'-Hidroxil-Quinasa/genética
3.
J Biol Chem ; 300(6): 107368, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38750793

RESUMEN

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.


Asunto(s)
Hidrolasas Diéster Fosfóricas , Humanos , Biología Computacional/métodos , Cristalografía por Rayos X , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Motivos de Unión al ARN/genética
4.
J Biol Chem ; 299(3): 102991, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36758800

RESUMEN

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.


Asunto(s)
Roturas del ADN de Doble Cadena , Escherichia coli , Animales , Humanos , Escherichia coli/genética , Reparación del ADN , Reparación del ADN por Unión de Extremidades , ADN , ARN/genética , ADN Ligasa (ATP) , Mamíferos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Enzimas Reparadoras del ADN/genética
5.
J Biol Chem ; 299(5): 104714, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37061005

RESUMEN

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.


Asunto(s)
Enzimas Reparadoras del ADN , Leucocitos Mononucleares , Fosfotransferasas (Aceptor de Grupo Alcohol) , Ataxias Espinocerebelosas , Humanos , ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ataxias Espinocerebelosas/genética
6.
Proc Natl Acad Sci U S A ; 117(14): 8154-8165, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32205441

RESUMEN

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.


Asunto(s)
Ataxina-3/genética , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Enfermedad de Machado-Joseph/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Anciano de 80 o más Años , Animales , Animales Modificados Genéticamente , Ataxina-3/metabolismo , Encéfalo/patología , Línea Celular , Roturas del ADN de Doble Cadena , Modelos Animales de Enfermedad , Drosophila , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Pluripotentes Inducidas , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Masculino , Ratones , Persona de Mediana Edad , Mutación , Péptidos/genética , ARN Interferente Pequeño/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(25): 14127-14138, 2020 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-32522879

RESUMEN

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.


Asunto(s)
Síndrome de Cockayne/genética , Proteínas de Unión al ADN/química , Endonucleasas/química , Proteínas Nucleares/química , Mutación Puntual , Factores de Transcripción/química , Xerodermia Pigmentosa/genética , Sitios de Unión , Secuencia Conservada , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Estabilidad de Enzimas , Humanos , Simulación de Dinámica Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
8.
Int J Mol Sci ; 24(18)2023 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-37762489

RESUMEN

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.


Asunto(s)
Medicina , Humanos , Animales , Ratones , Ratones Noqueados , Envejecimiento , Reparación del ADN , Biología
9.
Biophys J ; 120(15): 3152-3165, 2021 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-34197805

RESUMEN

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.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Modelos Moleculares , ARN Viral/genética , Dispersión del Ángulo Pequeño , Proteínas no Estructurales Virales , Replicación Viral , Difracción de Rayos X
10.
Nucleic Acids Res ; 46(9): 4515-4532, 2018 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-29522130

RESUMEN

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.


Asunto(s)
Transformación Celular Neoplásica , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Inestabilidad Genómica , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Línea Celular , Línea Celular Tumoral , Núcleo Celular/enzimología , Daño del ADN , Replicación del ADN , Desoxirribonucleasa (Dímero de Pirimidina)/genética , Células Epiteliales/enzimología , Humanos , Neoplasias Pulmonares/enzimología , Mutación , Mucosa Respiratoria/citología , Mucosa Respiratoria/enzimología
11.
Nucleic Acids Res ; 45(2): 739-748, 2017 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-27794043

RESUMEN

Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.


Asunto(s)
Factor 1 de Ensamblaje de la Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Oxidación-Reducción , Estrés Oxidativo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Daño del ADN/efectos de la radiación , ADN Glicosilasas/metabolismo , Glucosa Oxidasa/metabolismo , Histonas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos , Unión Proteica , Radiación Ionizante , Especies Reactivas de Oxígeno , Factores de Transcripción
12.
Clin Sci (Lond) ; 132(4): 475-488, 2018 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-29440622

RESUMEN

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.


Asunto(s)
Neoplasias Pulmonares/inducido químicamente , Fumar/efectos adversos , Factores de Tiempo , Contaminación por Humo de Tabaco/efectos adversos , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Incidencia , Ratones , Nicotiana/efectos adversos
13.
PLoS Genet ; 10(10): e1004686, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25299392

RESUMEN

As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.


Asunto(s)
Envejecimiento , Proteínas de Unión al ADN/deficiencia , Enfermedades Carenciales/etiología , Endonucleasas/deficiencia , Proteínas Nucleares/deficiencia , Factores de Transcripción/deficiencia , Envejecimiento/genética , Animales , Encéfalo/patología , Caquexia/etiología , Caquexia/genética , Sistema Nervioso Central/fisiología , Sistema Nervioso Central/fisiopatología , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Enfermedades Carenciales/genética , Modelos Animales de Enfermedad , Endonucleasas/genética , Endonucleasas/metabolismo , Femenino , Hígado/patología , Longevidad/genética , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoporosis/etiología , Osteoporosis/genética , Embarazo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
14.
J Biol Chem ; 290(41): 24636-48, 2015 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-26245904

RESUMEN

Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans.


Asunto(s)
ADN Glicosilasas/deficiencia , ADN Glicosilasas/genética , ADN/metabolismo , Genoma/genética , Transcripción Genética , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Línea Celular , ADN/genética , Daño del ADN , Técnicas de Inactivación de Genes , Inestabilidad Genómica , Homeostasis , Humanos , Inflamación/genética , Inflamación/metabolismo , Ratones , Oxidación-Reducción , ARN Polimerasa II/metabolismo , Telómero/genética
15.
Clin Sci (Lond) ; 130(14): 1269-84, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27129193

RESUMEN

Third hand smoke (THS) is the accumulation of second hand smoke (SHS) toxins on surfaces in homes, cars, clothing and hair of smokers. It is known that 88M US nonsmokers ≥3 years old living in homes of smokers are exposed to THS toxicants and show blood cotinine levels of ≥0.05 ng/ml, indicating that the toxins are circulating in their circulatory systems. The goal of the present study is to investigate the mechanisms by which THS causes impaired wound healing. We show that mice living under conditions that mimic THS exposure in humans display delayed wound closure, impaired collagen deposition, altered inflammatory response, decreased angiogenesis, microvessels with fibrin cuffs and a highly proteolytic wound environment. Moreover, THS-exposed mouse wounds have high levels of oxidative stress and significantly lower levels of antioxidant activity leading to molecular damage, including protein nitration, lipid peroxidation and DNA damage that contribute to tissue dysfunction. Furthermore, we show that elastase is elevated, suggesting that elastin is degraded and the plasticity of the wound tissue is decreased. Taken together, our results lead us to conclude that THS toxicants delay and impair wound healing by disrupting the sequential processes that lead to normal healing. In addition, the lack of elastin results in loss of wound plasticity, which may be responsible for reopening of wounds.


Asunto(s)
Nicotiana/efectos adversos , Contaminación por Humo de Tabaco/efectos adversos , Cicatrización de Heridas , Animales , Permeabilidad Capilar , Quimiocinas/biosíntesis , Colágeno/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Estrés Oxidativo , Inhibidor Tisular de Metaloproteinasa-1/análisis
16.
Mutagenesis ; 28(4): 381-91, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23462851

RESUMEN

Exposure to thirdhand smoke (THS) is a newly described health risk. Evidence supports its widespread presence in indoor environments. However, its genotoxic potential, a critical aspect in risk assessment, is virtually untested. An important characteristic of THS is its ability to undergo chemical transformations during aging periods, as demonstrated in a recent study showing that sorbed nicotine reacts with the indoor pollutant nitrous acid (HONO) to form tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-4-(3-pyridyl)butanal (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The goal of this study was to assess the genotoxicity of THS in human cell lines using two in vitro assays. THS was generated in laboratory systems that simulated short (acute)- and long (chronic)-term exposures. Analysis by liquid chromatography-tandem mass spectrometry quantified TSNAs and common tobacco alkaloids in extracts of THS that had sorbed onto cellulose substrates. Exposure of human HepG2 cells to either acute or chronic THS for 24h resulted in significant increases in DNA strand breaks in the alkaline Comet assay. Cell cultures exposed to NNA alone showed significantly higher levels of DNA damage in the same assay. NNA is absent in freshly emitted secondhand smoke, but it is the main TSNA formed in THS when nicotine reacts with HONO long after smoking takes place. The long amplicon-quantitative PCR assay quantified significantly higher levels of oxidative DNA damage in hypoxanthine phosphoribosyltransferase 1 (HPRT) and polymerase ß (POLB) genes of cultured human cells exposed to chronic THS for 24h compared with untreated cells, suggesting that THS exposure is related to increased oxidative stress and could be an important contributing factor in THS-mediated toxicity. The findings of this study demonstrate for the first time that exposure to THS is genotoxic in human cell lines.


Asunto(s)
Daño del ADN , Contaminación por Humo de Tabaco/efectos adversos , Línea Celular , Ensayo Cometa , Roturas del ADN/efectos de los fármacos , Humanos , Mutágenos/análisis , Mutágenos/química , Mutágenos/toxicidad , Ácido Nitroso/análisis , Ácido Nitroso/química , Ácido Nitroso/toxicidad , Estrés Oxidativo
17.
Methods Mol Biol ; 2701: 149-156, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37574480

RESUMEN

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.


Asunto(s)
Estructuras R-Loop , ARN , Humanos , Conformación de Ácido Nucleico , ARN/genética , ADN/genética , Anticuerpos/química , Inestabilidad Genómica
18.
bioRxiv ; 2023 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-37645927

RESUMEN

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.

19.
Nat Commun ; 14(1): 8169, 2023 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-38071370

RESUMEN

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.


Asunto(s)
COVID-19 , ADN Glicosilasas , Cricetinae , Animales , Humanos , COVID-19/genética , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Enzima Convertidora de Angiotensina 2/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Genoma , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo
20.
PLoS One ; 17(5): e0267839, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35576221

RESUMEN

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.


Asunto(s)
Nitrosaminas , Contaminación por Humo de Tabaco , Aldehídos , Carcinógenos/análisis , ADN , Daño del ADN , Inestabilidad Genómica , Humanos , Pulmón/metabolismo , Nicotina/análisis , Nitrosaminas/análisis , Nitrosaminas/toxicidad , Piridinas , ARN , Nicotiana/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Contaminación por Humo de Tabaco/análisis
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