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1.
Nat Commun ; 10(1): 2377, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147550

RESUMO

Glycans from microbial pathogens are well known pathogen-associated molecular patterns that are recognized by the host immunity; however, little is known about whether and how mammalian self-glycans activate the host immune response, especially in the context of autoimmune disease. Using biochemical fractionation and two-dimensional HPLC, we identify an abundant and bioactive free glycan, the Manß1-4GlcNAc disaccharide in TREX1-associated autoimmune diseases. We report that both monosaccharide residues and the ß1-4 linkage are critical for bioactivity of this disaccharide. We also show that Manß1-4GlcNAc is produced by oligosaccharyltransferase hydrolysis of lipid-linked oligosaccharides in the ER lumen, followed by ENGase and mannosidase processing in the cytosol and lysosomes. Furthermore, synthetic Manß1-4GlcNAc disaccharide stimulates a broad immune response in vitro, which is in part dependent on the STING-TBK1 pathway, and enhances antibody response in vivo. Together, our data identify Manß1-4GlcNAc as a novel innate immune modulator associated with chronic autoimmune diseases.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Dissacarídeos/imunologia , Imunidade Inata/imunologia , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Doenças Autoimunes/genética , Modelos Animais de Doenças , Retículo Endoplasmático , Exodesoxirribonucleases/genética , Fibroblastos , Camundongos , Fosfoproteínas/genética , Células RAW 264.7
2.
Talanta ; 202: 297-302, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171185

RESUMO

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5' end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5'-end and biotin modification at 3'-end). The formed blunt 5'-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research.


Assuntos
Técnicas Biossensoriais , Exodesoxirribonucleases/genética , Polarização de Fluorescência , MicroRNAs/genética , Nanosferas/química , Técnicas de Amplificação de Ácido Nucleico , Exodesoxirribonucleases/metabolismo , Humanos , MicroRNAs/metabolismo
3.
Tumour Biol ; 41(3): 1010428319830837, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30880589

RESUMO

The polymorphisms of invasion suppressor gene CDH1 and DNA mismatch repair gene Exo1 have been reported to play critical role in the development, tumorigenesis, and progression of several kinds of cancers including prostate cancer. This study was designed to analyze the contribution of single-nucleotide polymorphisms of the CDH1 (-160C/A) and Exo1 (K589E) to prostate cancer susceptibility in Bangladeshi population. The study included 100 prostate cancer cases and age-matched 100 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to determine the genetic polymorphisms. A significant association was found between CDH1 -160C/A (rs16260) and Exo1 (rs1047840, K589E) polymorphisms and prostate cancer risk. In case of CDH1 -160C/A polymorphism, the frequencies of the three genotypes C/C,C/A, and A/A were 45%, 48%, and 7% in cases and 63%, 32%, and 5% in controls, respectively. The heterozygote C/A genotype and combined C/A + A/A genotypes showed 2.10-fold (odds ratio = 2.1000, 95% confidence interval = 1.2956-4.0905, p = 0.013) and 2.08-fold (odds ratio = 2.0811, 95% confidence interval = 1.1820-3.6641, p = 0.011) increased risk of prostate cancer, respectively, when compared with homozygous C/C genotypes. The variant A allele also was associated with increased risk of prostate cancer (odds ratio = 1.6901, 95% confidence interval = 1.0740-2.6597, p = 0.0233). In case of Exo1 (K589E) polymorphism, G/A heterozygote, A/A homozygote, and combined G/A + A/A genotypes were found to be associated with 2.30-, 4.85-, and 3.04-fold higher risk of prostate cancer, respectively (odds ratio = 2.3021, 95% confidence interval = 2.956-4.0905, p = 0.0031; odds ratio = 4.8462, 95% confidence interval = 1.0198-23.0284, p = 0.0291; OR = 3.0362, 95% confidence interval = 1.7054-5.4053, p = 0.0001, respectively). The "A" allele showed significant association with increased susceptibility (2.29-fold) to prostate cancer (odds ratio = 2.2955, 95% confidence interval = 1.4529-3.6270, p = 0.0004). Our results suggest that CDH1 -160C/A and Exo1 K589E polymorphisms are associated with increased susceptibility to prostate cancer in Bangladeshi population.


Assuntos
Antígenos CD/genética , Caderinas/genética , Enzimas Reparadoras do DNA/genética , Grupos Étnicos/genética , Exodesoxirribonucleases/genética , Predisposição Genética para Doença/genética , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Próstata/genética , Idoso , Alelos , Bangladesh , Estudos de Casos e Controles , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade
4.
Nucleic Acids Res ; 47(7): 3550-3567, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30698745

RESUMO

Activation of the checkpoint protein Tel1 requires the Mre11-Rad50-Xrs2 (MRX) complex, which recruits Tel1 at DNA double-strand breaks (DSBs) through direct interaction between Tel1 and Xrs2. However, in vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role also for the MR subcomplex in Tel1 activation. Here we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1-MRX interaction leading to poor Tel1 association at DSBs and consequent loss of Tel1 activation. The Mre11-S499P variant reduces Mre11-Rad50 interaction, suggesting an important role for MR complex formation in Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly 'closed' conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11-Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP-bound conformational state.


Assuntos
Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ativação Transcricional/genética , Trifosfato de Adenosina/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA Fúngico/genética , Conformação Molecular , Complexos Multiproteicos/genética , Ligação Proteica/genética , Multimerização Proteica/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
5.
Nucleic Acids Res ; 47(4): 1950-1963, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30624736

RESUMO

Bacteriophage λ encodes a DNA recombination system that includes a 5'-3' exonuclease (λ Exo) and a single strand annealing protein (Redß). The two proteins form a complex that is thought to mediate loading of Redß directly onto the single-stranded 3'-overhang generated by λ Exo. Here, we present a 2.3 Å crystal structure of the λ Exo trimer bound to three copies of the Redß C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The Redß CTD forms a three-helix bundle with unexpected structural homology to phage λ Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Redß binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to λ Exo to facilitate loading of Redß directly onto the initial single-stranded DNA (ssDNA) at a 3'-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.


Assuntos
Bacteriófago lambda/enzimologia , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Exodesoxirribonucleases/química , Proteínas Virais/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Exodesoxirribonucleases/genética , Mutação/genética , Ligação Proteica , Domínios Proteicos , Recombinação Genética , Proteínas Virais/genética
7.
Nucleic Acids Res ; 47(4): 1847-1860, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30544222

RESUMO

Chromosome duplication initiates via the assembly of replication fork complexes at defined origins, from where they proceed in opposite directions until they fuse with a converging fork. Recent work highlights that the completion of DNA replication is highly complex in both pro- and eukaryotic cells. In this study we have investigated how 3' and 5' exonucleases contribute towards the successful termination of chromosome duplication in Escherichia coli. We show that the absence of 3' exonucleases can trigger levels of over-replication in the termination area robust enough to allow successful chromosome duplication in the absence of oriC firing. Over-replication is completely abolished if replication fork complexes are prevented from fusing by chromosome linearization. Our data strongly support the idea that 3' flaps are generated as replication fork complexes fuse. In the absence of 3' exonucleases, such as ExoI, these 3' flaps can be converted into 5' flaps, which are degraded by 5' exonucleases, such as ExoVII and RecJ. Our data support the idea that multiple protein activities are required to process fork fusion intermediates. They highlight the complexity of fork fusions and further support the idea that the termination area evolved to contain fork fusion-mediated pathologies.


Assuntos
Duplicação Cromossômica/genética , Replicação do DNA/genética , Escherichia coli/genética , Exonucleases/genética , Cromossomos Bacterianos/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Exodesoxirribonucleases/genética , Complexo de Reconhecimento de Origem/genética
8.
Nucleic Acids Res ; 47(4): 1814-1822, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30541106

RESUMO

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5' flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5' flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Exodesoxirribonucleases/genética , Endonucleases Flap/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , DNA/genética , Células Eucarióticas , Genoma Fúngico/genética , Saccharomyces cerevisiae/genética
9.
Genetics ; 211(2): 515-530, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30538107

RESUMO

The Mre11-Rad50-Xrs2 (MRX) complex acts together with the Sae2 protein to initiate resection of DNA double-strand breaks (DSBs) and to regulate a checkpoint response that couples cell cycle progression with DSB repair. Sae2 supports resistance to DNA damage and downregulates the signaling activities of MRX, Tel1, and Rad53 checkpoint proteins at the sites of damage. How these functions are connected to each other is not known. Here, we describe the separation-of-function sae2-ms mutant that, similar to SAE2 deletion, upregulates MRX and Tel1 signaling activities at DSBs by reducing Mre11 endonuclease activity. However, unlike SAE2 deletion, Sae2-ms causes neither DNA damage sensitivity nor enhanced Rad53 activation, indicating that DNA damage resistance depends mainly on Sae2-mediated Rad53 inhibition. The lack of Sae2, but not the presence of Sae2-ms, impairs long-range resection and increases both Rad9 accumulation at DSBs and Rad53-Rad9 interaction independently of Mre11 nuclease activity. Altogether, these data lead to a model whereby Sae2 plays distinct functions in limiting MRX-Tel1 and Rad9 abundance at DSBs, with the control on Rad9 association playing the major role in supporting DNA damage resistance and in regulating long-range resection and checkpoint activation.


Assuntos
Reparo do DNA , Endonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Quebras de DNA de Cadeia Dupla , Regulação para Baixo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Proc Natl Acad Sci U S A ; 115(38): 9557-9562, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30181269

RESUMO

Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc3Man9GlcNAc2-LLO but not Man9GlcNAc2-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST-dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST-dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.


Assuntos
Hexosiltransferases/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Técnicas de Inativação de Genes , Glicosilação , Hexosiltransferases/genética , Humanos , Hidrólise , Isoenzimas , Proteínas de Membrana/genética , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico/fisiologia
11.
Proc Natl Acad Sci U S A ; 115(35): 8793-8798, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104346

RESUMO

Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11-Rad50-Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre112-Rad502 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid.


Assuntos
Cromátides/metabolismo , Cromossomos Fúngicos/metabolismo , Reparo do DNA/fisiologia , DNA Fúngico/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Cromátides/genética , Cromossomos Fúngicos/genética , Replicação do DNA/fisiologia , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Complexos Multiproteicos/genética , Mutação , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
12.
Chem Commun (Camb) ; 54(75): 10562-10565, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30065993

RESUMO

Here, we introduced a novel exonuclease-assisted isothermal nucleic acid amplification (Exo-NAT) utilizing the full-length Bst DNA polymerase combined with a melting curve analysis. This method achieved an ultrahigh specificity with a good detection limit (102 copies) in both singleplex and multiplex detection, which was validated by detecting three diarrhea-inducing pathogens, rotavirus A, astrovirus, and adenovirus, in 42 clinical samples.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Genes Virais/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Adenoviridae/genética , Avastrovirus/genética , DNA Polimerase Dirigida por DNA/química , Exodesoxirribonucleases/química , Corantes Fluorescentes/química , Geobacillus stearothermophilus/enzimologia , Substâncias Intercalantes/química , Limite de Detecção , Compostos Orgânicos/química , Rotavirus/genética , Temperatura de Transição
13.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29976674

RESUMO

Over the past few decades, a large number of studies have identified herpesvirus sequences from many mammalian species around the world. Among the different nonhuman primate species tested so far for cytomegaloviruses (CMVs), only a few were from the New World. Seeking to identify CMV homologues in New World monkeys (NWMs), we carried out molecular screening of 244 blood DNA samples from 20 NWM species from Central and South America. Our aim was to reach a better understanding of their evolutionary processes within the Platyrrhini parvorder. Using PCR amplification with degenerate consensus primers targeting highly conserved amino acid motifs encoded by the herpesvirus DNA polymerase gene, we characterized novel viral sequences from 12 species belonging to seven genera representative of the three NWM families. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, and phylogenetic analyses confirmed that they all belonged to the Cytomegalovirus genus. Previously determined host taxa allowed us to demonstrate a good correlation between the distinct monophyletic clades of viruses and those of the infected primates at the genus level. In addition, the evolutionary branching points that separate NWM CMVs were congruent with the divergence dates of their hosts at the genus level. These results significantly expand our knowledge of the host range of this viral genus and strongly support the occurrence of cospeciation between these viruses and their hosts. In this respect, we propose that NWM CMV DNA polymerase gene sequences may serve as reliable molecular markers with which to infer Platyrrhini phylogenetics.IMPORTANCE Investigating evolutionary processes between viruses and nonhuman primates has led to the discovery of a large number of herpesviruses. No study published so far on primate cytomegaloviruses has extensively studied New World monkeys (NWMs) at the subspecies, species, genus, and family levels. The present study sought to identify cytomegalovirus homologues in NWMs and to decipher their evolutionary relationships. This led us to characterize novel viruses from 12 of the 20 primate species tested, which are representative of the three NWM families. The identification of distinct viruses in these primates not only significantly expands our knowledge of the host range of this viral genus but also sheds light on its evolutionary history. Phylogenetic analyses and molecular dating of the sequences obtained support a virus-host coevolution.


Assuntos
Citomegalovirus/classificação , Citomegalovirus/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Doenças dos Macacos/virologia , Filogenia , Platirrinos/virologia , Proteínas Virais/genética , Animais , América Central/epidemiologia , Citomegalovirus/enzimologia , DNA Viral/sangue , DNA Viral/genética , DNA Viral/isolamento & purificação , Evolução Molecular , Doenças dos Macacos/sangue , Doenças dos Macacos/epidemiologia , Reação em Cadeia da Polimerase/métodos , América do Sul/epidemiologia
14.
Rev Invest Clin ; 70(2): 68-75, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29718010

RESUMO

Background: Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset, autosomal dominant disease involving microvessels of the brain and eye resulting in central nervous system degeneration with visual disturbances, stroke, motor impairment, and cognitive decline. Frameshift mutations at the C-terminus of TREX1 gene are the molecular cause of this disorder. Objectives: The objective of this study is to present the different clinical manifestations of RVCL in three-related patients and to investigate the presence of TREX1 mutation in the extended genealogy. Methods: Multidisciplinary testing was performed in three related patients. Based on their family history, the study was extended to 34 relatives from the same small community. Neurological evaluation, sequencing of TREX1, and presymptomatic diagnosis were offered to all participants. Results: The patients exhibited the heterozygous TREX1 mutation p.V235Gfs*6, but with phenotypic variability. In addition, 15 relatives were identified as pre-manifest mutation carriers. The remaining participants did not carry the mutation. Conclusions: This is the figrst report of a large Mexican genealogy with RVCL, where the same TREX1 mutation causes a variation in organ involvement and clinical progression. The early identification and follow-up of individuals at risk may help provide insights into the basis for this variability in presentation.


Assuntos
Variação Biológica da População , Exodesoxirribonucleases/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Fosfoproteínas/genética , Doenças Retinianas/fisiopatologia , Doenças Vasculares/fisiopatologia , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Heterozigoto , Humanos , Masculino , México , Pessoa de Meia-Idade , Mutação , Doenças Retinianas/diagnóstico , Doenças Retinianas/genética , Doenças Vasculares/diagnóstico , Doenças Vasculares/genética
15.
Int J Mol Sci ; 19(4)2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29614055

RESUMO

Chromosomal interactions connect distant enhancers and promoters on the same chromosome, activating or repressing gene expression. PEAR1 encodes the Platelet-Endothelial Aggregation Receptor 1, a contact receptor involved in platelet function and megakaryocyte and endothelial cell proliferation. PEAR1 expression during megakaryocyte differentiation is controlled by DNA methylation at its first CpG island. We identified a PEAR1 cell-specific methylation sensitive region in endothelial cells and megakaryocytes that showed strong chromosomal interactions with ISGL20L2, RRNAD1, MRLP24, HDGF and PRCC, using available promoter capture Hi-C datasets. These genes are involved in ribosome processing, protein synthesis, cell cycle and cell proliferation. We next studied the methylation and expression profile of these five genes in Human Umbilical Vein Endothelial Cells (HUVECs) and megakaryocyte precursors. While cell-specific PEAR1 methylation corresponded to variability in expression for four out of five genes, no methylation change was observed in their promoter regions across cell types. Our data suggest that PEAR1 cell-type specific methylation changes may control long distance interactions with other genes. Further studies are needed to show whether such interaction data might be relevant for the genome-wide association data that showed a role for non-coding PEAR1 variants in the same region and platelet function, platelet count and cardiovascular risk.


Assuntos
Metilação de DNA , Receptores de Superfície Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ilhas de CpG , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Receptores de Superfície Celular/metabolismo
16.
Gene ; 665: 49-56, 2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-29705126

RESUMO

In the absence of the RNA-templated reverse transcriptase, telomerase, the predominant means of terminal addition, arises from break-induced replication (BIR) at multiple homologous subtelomeric Y' loci and among internal homeologous (imperfect) (polyG1-3T) tracts. These last tracts are interspersed between subtelomeric Y' direct repeats. One major survivor class contains very short (~50 bp) terminal telomere repeats. This size is sufficient for slow growth and partial telomere functionality and cell viability. However, in cells carrying the mre11A470T allele, adjacent to the predicted Rad50/Mre11 junction, cells thrive at wild-type rates, with small, but reproducible, increases in telomere length. We have proposed that the increase in telomere size and growth rate are causally linked. To understand the BIR process at the telomere, we initiated studies of large-tract (RAD51-sensitive) homologous BIR in MRE11 and mre11A470T cells in a model color assay coupled with CHEF gel analysis and marker retention. Wild-type and mutant homologous BIR rates are maintained at the same level as the rates between wild-type and mutant homeologous BIR. However, the fidelity of BIR products was severely altered in mre11A470T cells. We find that 95% of homologous BIR in MRE11 cells gives rise to the expected product size, while 25% of BIR products in mre11A470T cells were of unpredicted size (error-prone), most of which initiated at an aberrant site. However, ~25% of homeologous MRE11 cells and 1/7 of homeologous mre11A470T cells underwent error-prone BIR. This class is initiated erroneously, followed by secondary events that elongate or truncate the telomere. We conclude that error-prone BIRs are increased in homeologous recombination in wild-type and in mre11A470T cells. This finding may explain the bypass of senescence in telomerase-negative cells.


Assuntos
Replicação do DNA , DNA Fúngico , Endodesoxirribonucleases , Exodesoxirribonucleases , Mutação de Sentido Incorreto , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Substituição de Aminoácidos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Biosens Bioelectron ; 109: 190-196, 2018 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-29558733

RESUMO

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h1DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h1DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h2DNA and h3DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels.


Assuntos
Técnicas Biossensoriais , Catálise , DNA Viral/isolamento & purificação , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Fosfatase Alcalina/química , Condutometria , DNA Viral/química , DNA Viral/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Hibridização de Ácido Nucleico/genética
18.
PLoS Genet ; 14(3): e1007250, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29505562

RESUMO

Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO). Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs) selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.


Assuntos
Dano ao DNA/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Epistasia Genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genoma Fúngico , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Proc Natl Acad Sci U S A ; 115(13): E2921-E2929, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531047

RESUMO

Most replicative DNA polymerases (DNAPs) are endowed with a 3'-5' exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp121 of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp90 in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.


Assuntos
Ácido Aspártico/genética , Fagos Bacilares/enzimologia , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação , Sequência de Aminoácidos , Fagos Bacilares/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Mutagênese Sítio-Dirigida , Homologia de Sequência
20.
Funct Integr Genomics ; 18(4): 357-367, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29524012

RESUMO

The ubiquitous SbcCD exonuclease complex has been shown to perform an important role in DNA repair across prokaryotes and eukaryotes. However, they have remained uncharacterized in the ancient and stress-tolerant cyanobacteria. In the cyanobacterium Anabaena sp. strain PCC7120, SbcC and SbcD homologs, defined on the basis of the presence of corresponding functional domains, are annotated as hypothetical proteins, namely Alr3988 and All4463 respectively. Unlike the presence of sbcC and sbcD genes in a bicistronic operon in most organisms, these genes were distantly placed on the chromosome in Anabaena, and found to be negatively regulated by LexA. Both the genes were found to be essential in Anabaena as the individual deletion mutants were non-viable. On the other hand, the proteins could be individually overexpressed in Anabaena with no effect on normal cell physiology. However, they contributed positively to enhance the tolerance to different DNA damage-inducing stresses, such as mitomycin C and UV- and γ-radiation. This indicated that the two proteins, at least when overexpressed, could function independently and mitigate the damage caused due to the formation of DNA adducts and single- and double-strand breaks in Anabaena. This is the first report on possible independent in vivo functioning of SbcC and SbcD homologs in any bacteria, and the first effort to functionally characterize the proteins in any cyanobacteria.


Assuntos
Anabaena/genética , Proteínas de Bactérias/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Anabaena/efeitos dos fármacos , Anabaena/efeitos da radiação , Proteínas de Bactérias/genética , Adutos de DNA/genética , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Exodesoxirribonucleases/genética , Raios gama , Mitomicina/toxicidade , Raios Ultravioleta
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